Spinosyn-producing polyketide synthases

ABSTRACT

The invention provides, biologically active spinosyns, hybrid spinosyn polyketide synthases capable of functioning in  Saccharopolyspora spinosa  to produce the spinosyns, and methods of controlling insects using the spinosyns.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/073,226, filed Mar. 28, 2011 (now U.S. Pat. No. 8,624,009, issued onJan. 7, 2014). U.S. Pat. No. 8,624,009 is a divisional of U.S. patentapplication Ser. No. 12/616,470, filed Nov. 11, 2009, which is adivisional of U.S. patent application Ser. No. 11/254,686, filed Oct.20, 2005 (now U.S. Pat. No. 7,626,010, issued on Dec. 1, 2009. U.S. Pat.No. 7,626,010 is a divisional of U.S. patent application Ser. No.10/368,770, filed Feb. 19, 2003, which claims the benefit of U.S.Provisional Application Ser. No. 60/358,075, filed Feb. 19, 2002. Thedisclosure of each of the foregoing is hereby incorporated herein in itsentirety by this reference.

SUMMARY OF THE INVENTION

The present invention provides novel hybrid polyketide synthases (PKSs),DNA encoding such PKSs, vectors incorporating the hybrid polyketidesynthase DNA, host organisms including but not limited toSaccharopolyspora spinosa strains transformed with the hybrid polyketidesynthase DNA, methods of using the hybrid polyketide synthase DNA tochange the products made by spinosyn-producing strains, and the novelbiologically active compounds generated by these manipulations.

BACKGROUND OF THE INVENTION

As disclosed in U.S. Pat. No. 5,362,634, fermentation product A83543 isa family of related compounds produced by Saccharopolyspora spinosa. Thefamily of natural spinosyn compounds that have previously been isolatedare described in U.S. Pat. No. 6,274,350 B1 and WO 01/19840, along withtheir activities in a variety of insect control assays. A number ofsemi-synthetic spinosyn analogues are also described in U.S. Pat. No.6,001,981, in which the chemically accessible areas of the spinosynmolecule were successfully substituted in a variety of ways.

The known members of this family have been referred to as factors orcomponents, and each has been given an identifying letter designation.These compounds are hereinafter referred to as spinosyn A, B, etc. Thespinosyn compounds are useful for the control of arachnids, nematodesand insects, in particular, Lepidoptera and Diptera species, which arequite environmentally friendly and have an appealing toxicologicalprofile. The commercial product Spinosad is a mixture of spinosyns A andD (Pesticide Manual, 11th ed., p. 1272).

Tables 1 and 2 identify the structures of some known spinosyn compounds:

TABLE 1

Factor R1 R2 R3 R4 R5 R6 R7 spinosyn A H CH₃

C₂H₅ CH₃ CH₃ CH₃ spinosyn B H CH₃

C₂H₅ CH₃ CH₃ CH₃ spinosyn C H CH₃

C₂H₅ CH₃ CH₃ CH₃ spinosyn D CH₃ CH₃ (a) C₂H₅ CH₃ CH₃ CH₃ spinosyn E HCH₃ (a) CH₃ CH₃ CH₃ CH₃ spinosyn F H H (a) C₂H₅ CH₃ CH₃ CH₃ spinosyn A17-Psa H CH₃ H C₂H₅ CH₃ CH₃ CH₃ spinosyn D 17-Psa CH₃ CH₃ H C₂H₅ CH₃ CH₃CH₃ spinosyn E 17-Psa H CH₃ H CH₃ CH₃ CH₃ CH₃ spinosyn F 17-Psa H H HC₂H₅ CH₃ CH₃ CH₃

TABLE 2

Factor R1 R2 R3 R4 R5 spinosyn A 9-Psa H CH₃

C₂H₅ H spinosyn D 9-Psa CH₃ CH₃ (a) C₂H₅ H spinosyn A H CH₃ H C₂H₅ Haglycone spinosyn D CH₃ CH₃ H C₂H₅ H aglycone

The naturally produced spinosyn compounds consist of a 5,6,5-tricyclicring system, fused to a 12-membered macrocyclic lactone, a neutral sugar(rhamnose) and an amino sugar (forosamine) (see Kirst et al. (1991). Ifthe amino sugar is not present the compounds have been referred to asthe pseudoaglycone of A, D, etc., and if the neutral sugar is notpresent then the compounds have been referred to as the reversepseudoaglycone of A, D, etc. A more preferred nomenclature is to referto the pseudoaglycones as spinosyn A 17-Psa, spinosyn D 17-Psa, etc.,and to the reverse pseudoaglycones as spinosyn A 9-Psa, spinosyn D9-Psa, etc.

The naturally produced spinosyn compounds may be produced viafermentation from cultures NRRL 18395, 18537, 18538, 18539, 18719,18720, 18743 and 18823. These cultures have been deposited and made partof the stock culture collection of the Midwest Area Northern RegionalResearch Center, Agricultural Research Service, United States Departmentof Agriculture, 1815 North University Street, Peoria, Ill. 61604.

U.S. Pat. No. 5,362,634 and corresponding European Patent ApplicationNo. 375316 A1 disclose spinosyns A, B, C, D, E, F, G, H, and J. Thesecompounds are disclosed as being produced by culturing a strain of thenovel microorganism Saccharopolyspora spinosa selected from NRRL 18395,NRL 18537, NRRL 18538, and NRRL 18539.

WO 93/09126 disclosed spinosyns L, M, N, Q, R, S, and T. Also disclosedtherein are two spinosyn J producing strains: NRRL 18719 and NRRL 18720,and a strain that produces spinosyns Q, R, S, and T: NRRL 18823.

WO 94/20518 and U.S. Pat. No. 5,670,486 disclose spinosyns K, O, P, U,V, W, and Y, and derivatives thereof. Also disclosed is spinosynK-producing strain NRRL 18743.

WO 01/19840 discloses spinosyn analogs produced by culturingSaccharopolyspora species LW107129 (NRRL 30141).

WO 99/46387 and U.S. Pat. No. 6,143,526 disclose the spinosynbiosynthetic genes from Saccharopolyspora spinosa.

The nature of the genes involved in spinosyn biosynthesis, together withprevious studies of precursor incorporation (Broughton et al., 1991),indicate that spinosyns are produced by the stepwise condensation of2-carbon and 3-carbon carboxylic acids to generate a polyketide that iscyclized and bridged. The tetracyclic, aglycone product of thesereactions is converted to the pseudoaglycone by addition of a rhamnosylresidue, and synthesis is completed by the addition of thedi-N-methylated sugar, forosamine. In some aspects, this process issimilar to the biosynthetic pathway by which other macrolides (such asthe antibiotic erythromycin, the antihelmintic avermectin, and theimmunosuppressant rapamycin) are produced. In particular, the polyketidenucleus is assembled by a very large, multifunctional protein that is aType I polyketide synthase (spn PKS). This polypeptide complex comprisesa loading module and ten extension modules, each module beingresponsible for both the addition of a specific acyl-CoA precursor tothe growing polyketide chain, and for the degree of reduction of theβ-keto carbonyl group. Each module performs several biochemicalreactions that are carried out by specific domains of the polypeptide.All the extension modules contain an acyl transferase (AT) domain thatdonates the acyl group from a precursor to an acyl carrier protein (ACP)domain, and a β-ketosynthase (KS) domain that adds the pre-existingpolyketide chain to the new acyl-ACP by decarboxylative condensation.Additional domains are present in some extension modules:β-ketoreductase (KR) domains reduce β-keto groups to hydroxyls,dehydratase (DH) domains remove hydroxyls to leave double bonds, and theenoyl reductase (ER) domain reduces a double bond to leave a saturatedcarbon. The loading module of the spn PKS is different from theextension modules in that it contains a variant KS domain (KSq), as wellas AT and ACP domains. The KSq domain, which is also found in some otherType I PKS loading modules (but not all), is believed to provide therequisite starter unit by decarboxylation of an ACP-bound acyl chain(Bisang et al., 1999). The terminal extension module contains athioesterase/cyclase (TE) domain that liberates the polyketide chainfrom the PKS.

The spinosyn PKS DNA region consists of 5 ORFs with in-frame stop codonsat the end of some ACP domains, similar to the PKS ORFs in the othermacrolide-producing bacteria. The five spinosyn PKS genes are arrangedhead-to-tail, without any intervening non-PKS functions such as theinsertion element found between the erythromycin PKS genes AI and AII(Donadio et al., 1993). They are designated spnA, spnB, spnC, spnD, andspnE. The nucleotide sequence for each of the five spinosyn PKS genes,and the corresponding polypeptides, are identified in U.S. Pat. No.6,143,526 and in Waldron et al., 2001. Also identified in these sourcesare the predicted translation products of the PKS genes, and theboundaries of the domains and modules.

After it is synthesized, the spinosyn polyketide precursor condenses toform a macrocyclic lactone, referred to hereinafter as the polyketidenucleus. Production of insecticidally active spinosyns requiresadditional processing of the polyketide nucleus. First, carbon-carbonbridges must be formed between C3 and C14, C4 and C12, and C7 and C11,to generate the aglycone intermediate. Possible mechanisms for theseunusual reactions have been suggested (Waldron et al., 2001), but thestructural features of the polyketide substrate that are required forthem to occur are not known. Second, a tri-O-methyl rhamnose must beincorporated at C9 to generate the pseudoaglycone. It is not known ifthe rhamnose is normally methylated before or after its addition to theaglycone, but S. spinosa is capable of adding the methyl groups afterthe rhamnose moiety has been conjugated to the aglycone (Broughton etal., 1991). The methylations must occur in a particular sequence (2′then 3′ then 4′) or not all of them will take place, indicating that themethyltransferases have very specific substrate requirements. The thirdprocessing step, addition of forosamine at C17, is needed to produce themost active spinosyns. The enzymes involved in this step also havestringent substrate requirements: the forosaminyl transferase will notuse the aglycone as a substrate, and the N-methyltransferase will notact on the forosamine after it has been attached to the pseudoaglycone.This substrate-specificity of later biosynthetic enzymes may be abarrier to producing novel, biologically active spinosyns fromprecursors with different chemical structures.

In certain cases polyketide synthase (PKS) genes have previously beenmanipulated with the objective of providing novel polyketides. In-framedeletion of the DNA encoding part of the KR domain in module 5 of theerythromycin-producing (ery) PKS has been shown to lead to the formationof erythromycin analogues, namely5,6-dideoxy-3alpha-mycarosyl-5-oxoerythronolide B and5,6-dideoxy-5-oxoerythronolide B (Donadio et al., 1991). Likewise,alteration of active site residues in the ER domain of module 4 of theery PKS, by genetic engineering of the corresponding PKS-encoding DNAand its introduction into Saccharopolyspora erythraea, led to theproduction of 6,7-anhydroerythromycin C (Donadio et al., 1993). WO93/13663 describes additional types of genetic manipulation of the eryPKS genes that are capable of producing altered polyketides.

WO 98/01546 discloses replacement of the loading module of the ery PKSwith the loading module from the avennectin (ave) PKS, to produce ahybrid Type I PKS gene that incorporates different starter units to makenovel erythromycin analogues.

However, it has also been found that not all manipulations of PKS genesproduce the targeted new analogues. When Donadio et al. (1993)inactivated an ER domain of the ery PKS, the resultinganhydro-derivative could not be completely processed because it was nolonger a substrate for the mycarose-O-methyltransferase. Changing thepolyketide starter unit prevented complete elongation and elaboration ofa rifamycin analogue in Amycolatopsis mediterranei (Hunziker et al.,1998). Given the extensive substrate-specific processing that isrequired to generate insecticidally active spinosyns, it is not obviousthat genetic modifications that change the structure of a spinosynpolyketide will permit synthesis of a fully processed molecule withuseful biological activity. However, if such analogues could be made,and they had a different spectrum of insecticidal activity, they wouldbe highly desirable because known spinosyns do not control all pests.

BRIEF DESCRIPTION OF THE INVENTION

In one of its aspects, the invention provides a hybrid spinosynpolyketide synthase that is capable of functioning in Saccharopolysporaspinosa to produce a biologically active spinosyn, said hybridpolyketide synthase comprising a heterologous loading module operativelyassociated with a plurality of Saccharopolyspora spinosa extendermodules. In preferred embodiments, the spinosyn loading domain isreplaced with the loading domain for the erythromycin PKS or avermectinPKS. The ave and ery loading domains are of particular interest becausethey accept a variety of starter units. Also useful are hybrid PKS genesin which the heterologous loading module incorporates an unusual starterunit, such as the loading module for rapamycin (cyclohexene carboxylicacid) or for myxathiazole (3-methyl butyric acid). The requiredprecursors, e.g., cyclohexene carboxylic acid or 3-methyl butyric acid,may be provided in the culture medium, or the genes encoding theirbiosynthetic enzymes may be engineered into the organism so they aresynthesized endogenously.

In another of its aspects, the invention provides a hybrid spinosynpolyketide synthase that is capable of functioning in Saccharopolysporaspinosa to produce a biologically active 6-ethyl spinosyn compound,16-ethyl spinosyn compound, 18-ethyl spinosyn compound, or 20-ethylspinosyn compound, said hybrid polyketide synthase being the productproduced by spinosyn biosynthetic DNA that has been modified so that theDNA for the AT domain of module 8, 3, 2, or 1, respectively, in thespinosyn PKS is replaced with DNA for an AT domain that normallyincorporates ethyl malonyl-CoA. In preferred embodiments, the DNA thatencodes the relevant spinosyn AT domain is replaced with the DNA thatencodes the AT domain of module 5 of the tylosin PKS or module 5 of themonensin PKS. In preferred embodiments, the Streptomyces cinnamonensiscrotonyl-CoA reductase is co-expressed.

In another of its aspects, the invention provides a hybrid spinosynpolyketide synthase that is capable of functioning in Saccharopolysporaspinosa to produce a biologically active 18-methyl spinosyn compound, or20-methyl spinosyn compound, said hybrid polyketide synthase being theproduct produced by spinosyn biosynthetic DNA that has been modified sothat the DNA for the AT domain of module 2 or 1, respectively, in thespinosyn PKS is replaced with DNA for an AT domain that normallyincorporates methyl malonyl-CoA.

In another of its aspects, the invention provides a hybrid spinosynpolyketide synthase that is capable of functioning in Saccharopolysporaspinosa to produce a biologically active 16-desmethyl spinosyn compound,said hybrid polyketide synthase being the product produced by spinosynbiosynthetic DNA that has been modified so that the DNA for the ATdomain of module 3 in the spinosyn PKS is replaced with DNA for an ATdomain that normally incorporates malonyl-CoA. In a preferred embodimentthe AT domain of module 3 is replaced with the DNA that encodes the ATdomain of module 2 of the rapamycin PKS.

In another of its aspects, the invention provides a process forproducing a 6-ethyl spinosyn compound, a 21-desethyl-21-n-propylspinosyn compound, or a 6-ethyl-21-desethyl-21-propyl spinosyn compoundthat comprises culturing a transgenic host organism that coexpressescrotonyl-CoA reductase with the spinosyn biosynthetic pathway. In apreferred embodiment, the host organism is transformed with DNA encodingthe S. cinnamonensis crotonyl-CoA reductase.

In another of its aspects, the invention provides a Saccharopolysporaspinosa strain that has been transformed with DNA encoding the S.cinnamonensis crotonyl-CoA reductase.

In another of its aspects, the invention provides DNA encoding a hybridspinosyn polyketide synthase of the invention, as described above.

In another of its aspects, the invention provides a vector comprisingDNA as described above.

In another of its aspects, the invention provides a host organismcomprising DNA as described above.

In yet another of its aspects, the invention provides a compound of theformula (I)

wherein

-   -   R1 is hydrogen, methyl, or ethyl;    -   R2 is hydrogen, methyl, or ethyl;    -   R3 is hydrogen,

-   -   R4 is methyl or ethyl, either of which may be substituted with        one or more groups selected from halo, hydroxy, C₁-C₄ alkyl,        C₁-C₄ alkoxy, alkylthio, or cyano;    -   R4 is an alpha-branched C₃-C₅ alkyl group, C₃-C₈ cycloalkyl        group, or C₃-C₈ cycloalkenyl group, any of which may be        substituted with one or more groups selected from halo, hydroxy,        C₁-C₄ alkyl, C₁-C₄ alkoxy, alkylthio, or cyano; or    -   R4 is a 3-6 membered heterocyclic group that contains O or S,        that is saturated or fully or partially unsaturated, and that        may be substituted with one or more groups selected from halo,        hydroxy, C₁-C₄ alkyl, C₁-C₄ alkoxy, C₁-C₄ alkylthio, or cyano;    -   R5 is hydrogen or methyl;    -   R6 is hydrogen or methyl;    -   R7 is hydrogen or methyl;    -   R8 is hydrogen, methyl, or ethyl;    -   R9 is hydrogen, methyl, or ethyl;    -   or a 5,6-dihydro derivative of a compound of formula I,    -   provided that:        -   a) R4 is methyl or ethyl substituted with one or more groups            selected from halo, hydroxy, C₁-C₄ alkyl, C₁-C₄ alkoxy,            C₁-C₄ alkylthio, or cyano; or        -   b) R4 is an alpha-branched C₃-C₅ alkyl group, C₃-C₈            cycloalkyl group, or C₃-C₈ cycloalkenyl group, any of which            may be substituted with one or more groups selected from            halo, hydroxy, C₁-C₄ alkyl, C₁-C₄ alkoxy, C₁-C₄ alkylthio,            or cyano; or        -   c) R4 is a 3-6 membered heterocyclic group that contains O            or S, that is saturated or fully or partially unsaturated,            and that may be substituted with one or more groups selected            from halo, hydroxy, C₁-C₄ alkyl, C₁-C₄ alkoxy, C₁-C₄            alkylthio, or cyano; or        -   d) R1 or R2 is ethyl; or        -   e) R8 is methyl or ethyl; or        -   f) R9 is methyl or ethyl.

Illustrative compounds provided by the invention are identified in thefollowing Table 3. Compound numbers cited hereinafter refer to thecompounds identified in Table 3.

TABLE 3 (I)

cmpd. no. name R1 R2 R3 R4 R5 R6 R7 R8 R9  1 21-desethyl-21-cyclopropylspinosyn A H CH₃

cyclopropyl CH₃ CH₃ CH₃ H H  2 21-desethyl-21-cyclopropyl spinosyn D CH₃CH₃ (a) cyclopropyl CH₃ CH₃ CH₃ H H  3 21-desethyl-21-cyclopropylspinosyn A H CH₃ (a) cyclobutyl CH₃ CH₃ CH₃ H H  421-desethyl-21-cyclopropyl spinosyn D CH₃ CH₃ (a) cyclobutyl CH₃ CH₃ CH₃H H  5 21-desethyl-21-methythiomethyl H CH₃ (a) methylthio-methyl CH₃CH₃ CH₃ H H spinosyn A  6 21-desethyl-21-methylthiomethyl CH₃ CH₃ (a)methylthio-methyl CH₃ CH₃ CH₃ H H spinosyn D  721-desethyl-21-cyanomethyl spinosyn A H CH₃ (a) cyanomethyl CH₃ CH₃ CH₃H H  8 5,6-dihydro-21-desethyl-21-cyclobutyl H CH₃ (a) cyclobutyl CH₃CH₃ CH₃ H H spinosyn A  9 21-desethyl-21-isopropyl spinosyn A H CH₃ (a)isopropyl CH₃ CH₃ CH₃ H H 10 21-desethyl-21-isopropyl spinosyn D CH₃ CH₃(a) isopropyl CH₃ CH₃ CH₃ H H 11 21-desethyl-21-sec-butyl spinosyn A HCH₃ (a) sec-butyl CH₃ CH₃ CH₃ H H 12 21-desethyl-21-sec-butyl spinosyn DCH₃ CH₃ (a) sec-butyl CH₃ CH₃ CH₃ H H 1321-desethyl-21-methylcyclopropyl H CH₃ (a) methylcyclo-propyl CH₃ CH₃CH₃ H H spinosyn A 14 21-desethyl-21-methylcyclopropyl CH₃ CH₃ (a)methylcyclo-propyl CH₃ CH₃ CH₃ H H spinosyn D 1521-desethyl-21-(3-furyl) spinosyn A H CH₃ (a) 3-furyl CH₃ CH₃ CH₃ H H 1621-desethyl-21-(3-furyl) spinosyn D CH₃ CH₃ (a) 3-furyl CH₃ CH₃ CH₃ H H17 21-desethyl-21-cyclopropyl spinosyn A H CH₃ H cyclopropyl CH₃ CH₃ CH₃H H 17-pseudoaglycone 18 21-desethyl-21-cyclopropyl spinosyn D CH₃ CH₃ Hcyclopropyl CH₃ CH₃ CH₃ H H 17-pseudoaglycone 1921-desethyl-21-cyclobutyl spinosyn A H CH₃ H cyclobutyl CH₃ CH₃ CH₃ H H17-pseudoaglycone 20 21-desethyl-21-cyclobutyl spinosyn D CH₃ CH₃ Hcyclobutyl CH₃ CH₃ CH₃ H H 17-pseudoaglycone 21 16-desmethyl spinosyn DCH₃ H (a) ethyl CH₃ CH₃ CH₃ H H 22 16-desmethyl spinosyn D 17- CH₃ H Hethyl CH₃ CH₃ CH₃ H H pseudoaglycone 23 21-desethyl-21-n-propyl spinosynA H CH₃ (a) n-propyl CH₃ CH₃ CH₃ H H 24 6-ethyl spinosyn A C₂H₅ CH₃ (a)ethyl CH₃ CH₃ CH₃ H H 25 6-ethyl-21-desethyl-21-n-propyl C₂H₅ CH₃ (a)n-propyl CH₃ CH₃ CH₃ H H spinosyn A 26 16-desmethyl-16-ethyl spinosyn AH C₂H₅ (a) ethyl CH₃ CH₃ CH₃ H H 27 16-desmethyl-16-ethyl spinosyn D CH₃C₂H₅ (a) ethyl CH₃ CH₃ CH₃ H H 28 21-desethyl-21-n-propyl spinosyn D CH₃CH₃ (a) n-propyl CH₃ CH₃ CH₃ H H 29 5,6-dihydro-21-desethyl-21-n-propylH CH₃ (a) n-propyl CH₃ CH₃ CH₃ H H spinosyn A

The 5,6-dihydro derivatives of the compounds of Formula I (e.g.,compound 8 in Table 3) are compounds of the Formula II:

wherein R1, R2, R3, R4, R5, R6, R7, R8, and R9 are as defined forFormula I.

In another of its aspects, the invention provides a biologically pureculture of Saccharopolyspora spinosa selected from

-   -   NRRL 30539,    -   NRRL 30540,    -   NRRL 30541, and    -   NRRL 30542.

In another of its aspects, the invention provides a method ofcontrolling pests that comprises delivering to a pest an effectiveamount of a compound of claim 1.

In another of its aspects, the invention provides a pesticidecomposition comprising an effective amount of a compound of claim 1 asactive ingredient in combination with an appropriate diluent or carrier.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the construction of pCJR81.

FIG. 2 shows the construction of vectors pLSB2 and pLSB3 used forexpression of genes in S. spinosa.

FIG. 3 shows the construction of pLSB62, a vector to introduce the eryload into the spinosyn pathway.

FIG. 4 shows the hybrid ery/spn PKS pathway of S. spinosa 13E.

FIG. 5 shows the construction of pLSB29, a vector to introduce the aveload into the spinosyn pathway.

FIG. 6 shows the hybrid ave/spn PKS pathway of S. spinosa 21K2.

FIGS. 7 a and 7 b show the construction of pALK26, intermediate plasmidused to generate module 3 AT swap plasmids.

FIG. 8 shows the construction of pALK39, a vector used to introduce therapAT2 into spinosyn module 3.

FIG. 9 shows the construction of pALK21, a vector designed to expressthe S. cinnamonensis crotonyl-CoA reductase gene in S. spinosa.

FIG. 10 shows the construction of pALK36, a vector used to introduce thetylAT5 into spinosyn module 3.

FIG. 11 shows the hybrid PKS pathway of strain S. spinosa 7D23.

FIG. 12 shows the hybrid PKS pathway of strain S. spinosa 36P4.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is oligo PRIS1.

SEQ ID NO:2 is oligo PRIS2.

SEQ ID NO:3 is the DNA sequence of the promoter for resistance topristinamycin.

SEQ ID NO:4 is oligo CR311.

SEQ ID NO:5 is oligo CR312.

SEQ ID NO:6 is oligo SP28.

SEQ ID NO:7 is oligo SP29.

SEQ ID NO:8 is the fragment of DNA encoding the ery PKS loading modulethat was used for cloning, with introduced restriction enzyme sites atby 1-6 and by 1680-1685.

SEQ ID NO:9 is oligo SP14.

SEQ ID NO:10 is oligo SP15.

SEQ ID NO:11 is the fragment of DNA encoding the ave PKS loading modulethat was used for cloning, with introduced restriction enzyme sites atby 1-6 and by 1689-1694.

SEQ ID NO:12 is oligo CR322.

SEQ ID NO:13 is oligo CR323.

SEQ ID NO:14 is oligo CR324.

SEQ ID NO:15 is oligo CR325.

SEQ ID NO:16 is oligo CR328.

SEQ ID NO:17 is oligo CR329.

SEQ ID NO:18 is oligo CR330.

SEQ ID NO:19 is oligo CR321.

SEQ ID NO:20 is the fragment of DNA encoding the rap AT2 that was usedfor cloning, with introduced restriction enzyme sites at by 1-6 and by832-837.

SEQ ID NO:21 is oligo CCRMONF.

SEQ ID NO:22 is oligo CCRMONR.

SEQ ID NO:23 is the DNA sequence used for expression of the Streptomycescinnamonensis crotonyl-CoA reductase, with introduced restriction sitesat by 1-6 and 1389-1394.

SEQ ID NO:24 is oligo AK1.

SEQ ID NO:25 is oligo AK2.

SEQ ID NO:26 is the fragment of DNA encoding the tyl AT5 that was usedfor cloning, with introduced restriction enzyme sites at by 1-6 and by967-972.

DETAILED DESCRIPTION OF THE INVENTION

Culture Description

The novel strains derived from Saccharopolyspora spinosa NRRL 18537 orSaccharopolyspora spinosa NRRL 18538 and producing the compounds of theinvention are identified in the table below. The cultures have beendeposited in accordance with the terms of the Budapest treaty at theMidwest Area Regional Center, Agricultural Research Service, UnitedStates Department of Agriculture, 815 North University Street, Peoria,Ill. 61604. The strains were deposited on Jan. 15, 2001, and assignedthe deposit numbers as detailed in Table 4 below.

TABLE 4 Deposit Number Strain Parent Strain NRRL 30539 Saccharopolysporaspinosa 7D23 NRRL 18538 NRRL 30540 Saccharopolyspora spinosa 13E NRRL18537 NRRL 30541 Saccharopolyspora spinosa 21K2 NRRL 18538 NRRL 30542Saccharopolyspora spinosa 36P4 NRRL 18538Culture Characteristics

The novel strains derived from Saccharopolyspora spinosa NRRL 18537 orSaccharopolyspora spinosa NRRL 18538 and producing the compounds of theinvention had the following culture characteristics:

All cultures grew well on ISP2 and Bennett's agar and aerial hyphae wereproduced on all media used. The aerial spore mass was predominatelywhite (developing a pale pink hue with age). Substrate mycelium wascream to light tan in color, no distinctive pigment was apparent. Asoluble brown pigment was released into the medium. No significantdifferences were observed on any of the media used.

Manipulation of Spinosyn Pathway and Accessory Genes

In order to facilitate the manipulation of the spinosyn biosyntheticpathway, cosmids pRHB9A6 and pRHB3E11 were obtained and are described inU.S. Pat. No. 6,274,350 B1 and Waldron et al. (2001).

It is not directly apparent from the sequence of the spinosynbiosynthetic genes how production of spinosyns is regulated. In order toavoid potential complications associated with regulation of the genecluster, a number of heterologous promoters were used to drive all orpart of the polyketide synthase portion (genes spnA, spnB, spnC, spnDand spnE). The following promoters were found to be at least asefficient in the production of spinosyn PKS as the natural promoter(judged by production of the final products spinosyn A, spinosyn D,spinosyn A C17-pseudoaglycone, and spinosyn D C17-pseudoaglycone): Theacts promoter from the actinorhodin biosynthetic cluster of S.coelicolor, used along with its cognate activator, actII-ORF4, (asdescribed in WO 98/01546, WO 98/01571, Rowe et al., 1998) and; Thepromoter for the resistance to pristinamycin. The latter has previouslybeen reported to drive overexpression of polyketide genes from S.erythraea (Blanc et al., 1995; Sala-Bey et al., 1995). It is a promoterthat can be induced by physiological stresses in Streptomyces spp. Theuse of heterologous promoters in S. spinosa is not limited to thosedescribed above, and could include others that might be expected tofunction in S. spinosa.

Hybrid Spinosyn PKS Using Heterologous Loading Module

In one of its aspects the invention provides hybrid PKSs that arefunctional in Saccharopolyspora spinosa to generate polyketides that areprocessed to biologically active spinosyns. The resulting polyketideswere extended to the same length as the natural spinosyns, and processedby cross-bridging and glycosylation to generate novel insecticidalspinosyns. Preferably the hybrid PKS comprises the extension modulesfrom the spn PKS with a heterologous loading module that leads to aspinosyn polyketide having a different starter unit. Hybrid spinosyn PKSgenes that contain the spn extender modules behind a heterologousloading module can provide novel spinosyns with different side chains atC21. The nature of this side chain is determined by the starter unitthat the loading module selects to initiate polyketide synthesis.Changes in the side chain may alter the physical properties orbiological activity of the resulting spinosyn. It is particularly usefulto provide a hybrid PKS gene in which the loading module accepts manydifferent carboxylic acids, including unnatural acids. Such a gene canbe used to generate many different spinosyns by incorporation ofdifferent starter units. For example, the loading module of the spn PKScan be replaced by the loading module of the ave PKS, which is known toaccept a wide variety of starter units (Dutton et al., 1991). Theloading module of the ery PKS is also known to accept alternate starterunits (Pacey et al., 1998). Thus, an organism expressing such hybridspinosyn PKS genes can produce novel spinosyns in which the nature ofthe side chain at C21 is determined by the carboxylic acid that is fedto the organism. The side chains can be of different lengths, withbranches or cycles, and/or contain heteroatoms.

More preferably, the hybrid PKS includes a loading module that acceptsmany different carboxylic acids so the hybrid gene assembly can be usedto produce many different spinosyns. Particularly useful examplescontain the spn extension modules with the loading module from theerythromycin (ery) PKS, or the loading module from the avennectin (ave)PKS.

a. Hybrid Spinosyn PKS Using Ery Loading Module

The loading module of the erythromycin biosynthetic cluster (eryAT0ACP0)governs the introduction of propionyl-CoA into the starter of theerythromycin molecule. It has been shown previously that alternativestarters can be incorporated into the erythromycin molecule by feedingfree acids to the production medium (Pacey et al., 1998).

As shown in the following Examples, generation of a hybrid spnA gene inwhich the erythromycin loading module replaces the spinosyn loadingmodule leads to a spinosyn PKS that can accept free acids into thestarter unit. In the following illustrative Example, the erythromycinloading module was spliced to the beginning of the spinosyn KS1 justwithin the KS domain in the conserved region. New domain or moduleconnections are preferably made at conserved DNA sequences withindomains, or close to the edges thereof; however, an active polyketidesynthase can alternatively be generated by engineering splice sites inthe interdomain regions (WO 98/01546).

The ery load fragment was cloned in-frame with, and upstream of, aregion of spnA to allow homologous recombination with the native spnPKS. Upstream of the ery load was either the actI promoter (P_(actI)) orthe promoter for resistance to pristinamycin (P_(ptr)), giving rise tothe plasmids pLSB61 and pLSB62, respectively. These plasmids are basedon pKC1132 and are, therefore, apramycin-resistant. They also carry oriTfor conjugal transfer of DNA into actinomycetes (Bierman et al., 1992,Matsushima et al., 1994). These constructs were transformed into S.spinosa NRRL 18537 by conjugation. Exconjugants were confirmed tocontain the hybrid ery/spn PKS under the appropriate promoter by PCRamplification.

S. spinosa NRRL 18537:pLSB62 was designated S. spinosa 13E, and was usedfor analysis of spinosyn production.

As demonstrated in the following Examples, when cultured in productionmedia, S. spinosa 13E produced mainly spinosyn A and spinosyn E, inapproximately equal amounts. The total yield of spinosyns was estimatedto be approximately 10-25% of the wild-type spinosyn A levels, whichrepresented an increase in yield of spinosyn E of approximately 10-fold.This altered ratio of products may be a reflection of looser specificityof the ery loading module relative to the spn loading module, or areflection of the different substrate supply in S. spinosa compared withS. erythraea, or a combination of both. Incorporation of acetate by theery load has been observed in S. erythraea when the erythromycin pathwayis up-regulated (Rowe et al., 1998). The increase in yield of spinosyn Ein strain 13E over wild-type levels means that this would be a preferredstrain for the production of spinosyn E.

A number of carboxylic acids were fed to S. spinosa 13E, leading to theproduction of novel spinosyn analogues with altered starter units. Amongthe novel spinosyns identified were 21-desethyl-21-cyclopropyl spinosynsA and D (by incorporation of cyclopropane carboxylic acid),21-desethyl-21-cyclobutyl spinosyns A and D (by incorporation ofcyclobutane carboxylic acid) and 21-desethyl-21-methylthiomethylspinosyns A and D (from methylthioacetic acid). The novel analoguesshowed reasonable chromatographic retention times, characteristic UVchromophores and MS fragmentation patterns, and the predicted structureswere supported by accurate mass measurements. Structural assignments ofthe isolated 21-cyclopropyl and 21-cyclobutyl compounds were confirmedby full NMR characterization. Compounds isolated were active in insectcontrol assays.

The use of strain S. spinosa 13E is not limited to the production ofthese compounds. It is expected that a number of other spinosynanalogues can be identified by feeding other acids, such as those usedto produce novel erythromycins (Pacey et al., 1998).

EXAMPLE 1 Construction of pCJR81

See FIG. 1. Plasmid pCJR81 is a vector for expression of polyketidegenes under the promoter for resistance to pristinamycin. It wasconstructed as follows:

Two overlapping oligos were designed to perform a PCR reaction in whichthey act both as primers and template. They were designed to introducean NdeI restriction site incorporating the ATG start codon, such thatgenes can be cloned with optimal spacing from the ribosome binding site.A SpeI restriction site was incorporated to facilitate further cloning.The oligos are PRIS1 (SEQ ID NO:1) and PRIS2 (SEQ ID NO:2).

Amplification to obtain the promoter fragment was performed with Pwothermostable DNA polymerase using the manufacturer's conditions. The 112bp fragment was phosphorylated with T4 polynucleotide kinase, and clonedinto commercially available pUC18 digested with SmaI anddephosphorylated. Plasmids containing inserts were sequenced. Oneplasmid containing the correct sequence was designated pRIS4.

The 94 bp insert from pRIS4 was excised as a SpeI/NdeI fragment (SEQ IDNO:3) and cloned into pCJR24 (WO 98/01546, WO 98/01571, Rowe et al.,1998), which had been previously digested with SpeI and NdeI. Onecorrect plasmid was designated pCJR81.

EXAMPLE 2 Construction of Plasmids for Expression from S. Spinosad

See FIG. 2. Plasmids pLSB2 and pLSB3 were constructed for expression ofpolyketide genes or accessory genes in S. spinosa. Plasmid pLSB2contains the actI promoter (P_(actI)) and its cognate activator,actII-ORF4. Plasmid pLSB3 contains the promoter for resistance topristinamycin (P_(ptr)). These plasmids were constructed as follows:

Plasmid pKC1132 (Bierman et al., 1992) contains an origin of transfer(oriT), and an apramycin resistance marker for selection in both E. coliand actinomycetes. It can, therefore, be used for DNA manipulations inE. coli, and permit the final plasmids to be introduced into S. spinosaby conjugation. The polylinker of pKC1132 was replaced by a linker oftwo oligos CR311 (SEQ ID NO:4) and CR312 (SEQ ID NO:5) in order toprovide appropriate EcoRV/SpeI/NdeI/XbaI restriction sites.

Plasmid pKC1132 was digested with PvuII and the ends dephosphorylatedwith shrimp alkaline phosphatase. The oligos CR311 and CR312 werephosphorylated with T4 polynucleotide kinase, annealed and cloned intopKC1132_PvuII to generate pLSB1. A SpeI/NdeI fragment containing theactinorhodin pathway specific activator, actII-ORF4 and the actIpromoter was isolated from pCJR24 (WO 98/01546, WO 98/01571, Rowe etal., 1998) and a SpeI/NdeI fragment containing the promoter forresistance to pristinamycin was isolated from pCJR81 (described above,Example 1). Each of these fragments was cloned independently into pLSB1digested with SpeI and NdeI to generate pLSB2 (containing actII-ORF4 andP_(actI)) and pLSB3 (containing P_(ptr)).

EXAMPLE 3 Construction of a Vector to Incorporate the Loading Module ofthe Erythromycin Polyketide Synthase into the Spinosyn PolyketideSynthase

See FIG. 3. The vector to incorporate the loading module of theerythromycin polyketide synthase into the spinosyn polyketide synthasecontains the erythromycin loading module (AT0ACP0), followed by a regionof the first module of the spinosyn PKS to provide homology forintegration. The vector is designated pLSB62 and was constructed asfollows.

The erythromycin loading module was amplified by PCR using pCJR26 (Roweet al., 1998) as the template, and oligos SP28 (SEQ ID NO:6) and SP29(SEQ ID NO:7). SP28 incorporates an NdeI site at the start codon of theery sequence, and SP29 incorporates an NheI site at the beginning of theKS1 domain.

The PCR fragment was phosphorylated, gel-purified and cloned into pUC18,which had been previously digested with SmaI and dephosphorylated.Clones were screened for the presence of inserts and sequenced. Oneclone containing the correct sequence was designated pLSB44. Itcontained the insert in the orientation with the NheI site close to theEcoRI site of the polylinker. The sequence of the erythromycin loadingmodule fragment used, from the NdeI site to the NheI site is shown inSEQ ID NO:8.

Plasmid pLSB8 (described in Example 11) contains a fragment of spnAstarting with the NheI site at spnKS1. The fragment containing theerythromycin loading module was excised from pLSB44 as an NdeI/NheIfragment and cloned into pLSB8 previously digested with NdeI and NheI,to give pLSB56. The fragment contained in pLSB56 contains theerythromycin loading module spliced in-frame to the spinosyn KS1, with aregion of homology to spnA, which is sufficient for integration tooccur.

This region was removed as an NdeI/XbaI fragment and cloned into pLSB3to give pLSB62. This places the new ery/spn hybrid fragment underP_(ptr), in a vector that can be transferred into S. spinosa byconjugation and selected using the apramycin resistance marker.

EXAMPLE 4 Generation of a S. Spinosa Strain Harboring a HybridPolyketide Synthase Comprising the Erythromycin Loading Module Fused tothe KS1 of the Spinosyn PKS

See FIG. 4. Saccharopolyspora spinosa NRRL 18537 was transformed byconjugation (Matsushima et al., 1994) from E. coli S17-1 (Simon et al.,1983) with pLSB62. Transformants were selected for apramycin resistanceand screened by PCR. A single transformant was designated strain S.spinosa 13E.

EXAMPLE 5 Chemical Analysis of S. Spinosa Fermentations

The following HPLC method is useful for analyzing fermentations for theproduction of natural spinosyns and novel non-natural engineeredspinosyns.

In a 2 mL Eppendorf tube, an aliquot of fermentation broth (1 mL) wasadjusted to pH˜10 by the addition of 20% ammonia solution (ca. 20 μl).Ethyl acetate (1 mL) was added to the sample and mixed vigorously for 60seconds using a vortex. The mixture was separated by centrifugation in amicrofuge and the upper phase removed to a clean 2 mL Eppendorf tube.The ethyl acetate was removed by evaporation using a Speed-vac. Residueswere dissolved into methanol (250 μl) and clarified using a microfuge.Analysis was by the following HPLC system:

-   -   Injection volume: 50 μl    -   Column stationary phase: 150×4.6 mm column, base-deactivated        silica gel 3 μm (Hypersil C₁₈-BDS).    -   Mobile phase A: 10% acetonitrile: 90% water, containing 10 mM        ammonium acetate and 0.15% formic acid.    -   Mobile phase B: 90% acetonitrile: 10% water, containing 10 mM        ammonium acetate and 0.15% formic acid.    -   Mobile phase gradient: T=0 minute, 10% B; T=1, 10% B; T=25, 95%        B; T=29, 95% B; T=29.5, 10% B.    -   Flow rate: 1 mL/minute.    -   Detection: UV at 254 nm; MS over m/z range 100-1000.

EXAMPLE 6 Production of Metabolites by S. Spinosa 13E Fermentation

S. spinosa 13E was cultured from a frozen vegetative stock (1:1 CSMculture:cryopreservative, where the cryopreservative is 10% lactose, 20%glycerol w/v in water). A primary pre-culture was grown in CSM medium(tryptic soy broth 30 g/l, yeast extract 3 g/l, magnesium sulfate 2 g/l,glucose 5 g/l, maltose 4 g/l; Hosted and Baltz 1996; U.S. Pat. No.5,362,634), in a 50 mL culture in a 250 mL Erlenmeyer flask with a steelspring, shaken at 250 rpm with a two-inch throw at 30° C., 75% relativehumidity for 3 days. This was used to inoculate a secondary pre-culturein vegetative medium (glucose 10 g/l, N—Z-amine A 30 g/l, yeast extract3 g/l, magnesium sulfate 2 g/l; Strobel and Nakatsukasa 1993; U.S. Pat.No. 5,362,634) at 5% v/v, which was cultured under the same conditionsfor a further 2 days. The secondary vegetative pre-culture was used toinoculate production medium (glucose 67 g/l, Proflo cottonseed flour 25g/l, peptonized milk nutrient 22 g/l, corn steep liquor 12 g/l, methyloleate 40 g/l, calcium carbonate 5 g/l, pH to 7.0 with sodium hydroxide;Strobel and Nakatsukasa 1993) at 5% v/v. Small scale production cultureswere fermented under the same conditions as the pre-cultures, but for7-10 days. For initial S. spinosa 13E production, small-scale cultureswere grown in 30 mL of production medium in 250 mL Erlenmeyer flaskswith springs for 7 days.

For the identification of metabolites produced, a 1 mL aliquot offermentation broth was analyzed by LC-MS as described in Example 5. Bycomparison to authentic standards, and to a fermentation extract fromnon-transformed strain S. spinosa NRRL 18537, the major compoundsproduced by S. spinosa 13E were identified as spinosyns A and E (whichare produced in approximately equal amounts) and spinosyn D (which wasobserved as a minor product). The titer of strain S. spinosa 13E was˜10-15 mg/l of total spinosyns. The ratio of products for S. spinosa 13Ewas different to NRRL 18537, with the relative production of spinosyn Ebeing significantly increased.

EXAMPLE 7 Precursor-Directed Production of Novel Spinosyns from S.Spinosa 13E (Production of Compounds 1-6)

The ery/spn hybrid PKS was used to generate novel spinosyn metabolitesby feeding carboxylic acids to production cultures. The ery loadingmodule incorporated the carboxylic acid within the starter of themolecule.

Parallel production flasks (30 mL in 250 mL Erlenmeyer flask withspring) were inoculated as described in Example 6 above. After 24 hours,each of these was fed with a carboxylic acid (stock solutions made inwater and pH adjusted to 6.5 with sodium hydroxide) at a finalconcentration of 2-6 mM. After 7 days a 1 mL aliquot of fermentationbroth was analyzed by LC-MS as described in Example 5. The incorporationof cyclobutyl carboxylic acid, cyclopropyl carboxylic acid andmethylthioacetic acid to generate novel C21 modified spinosyns wasindicated by the appearance of novel peaks in the UV and MSchromatograms (Table 5). The MS spectra of the novel compounds gave ionsfor the [M+H]⁺ species and for the forosamine fragment (142.3).

TABLE 5 Compound Retention No. (see time Key Mass Spectral Carboxylicacid fed Table 3) (min) data (m/z) cyclopropyl carboxylic 1 23.5 744.4[M + H]⁺; 142.4 acid cyclopropyl carboxylic 2 25.0 758.5 [M + H]⁺; 142.3acid cyclobutyl carboxylic 3 25.7 758.5 [M + H]⁺; 142.3 acid cyclobutylcarboxylic 4 27.3 772.5 [M + H]⁺; 142.2 acid methylthio acetic acid 522.9 764.4 [M + H]⁺; 142.3 methylthio acetic acid 6 24.3 778.5 [M + H]⁺;142.3

EXAMPLE 8 Production and Isolation of 21-Desethyl-21-CyclobutylSpinosyns A and D (Compounds 3 and 4)

Frozen vegetative stocks of S. spinosa 13E were inoculated into primaryvegetative pre-cultures in CSM (50 mL incubated in a 250 mL Erlenmeyerflask with spring). Secondary pre-cultures in vegetative medium (250 mLincubated in a 2 L Erlenmeyer flask with spring) were prepared andincubated as described in Example 6, but at 300 rpm with a one-inchthrow.

Twelve to 14 L of production medium was prepared, as in Example 6, withthe addition of 0.01% v/v Pluronic L-0101 (BASF) antifoam. Productionmedium was inoculated with the secondary vegetative pre-culture at 5%v/v, and allowed to ferment in a 20 L stirred bioreactor for 7-10 daysat 30° C. Airflow was set at 0.75 vvm, over pressure was set at 0.5 bargor below, and impeller tip speed was controlled between 0.39 and 1.57ms⁻¹ in order to maintain dissolved oxygen tension at or above 30% ofair saturation. Additional Pluronic L0101 (BASF) was added to controlfoaming, if needed. Cyclobutyl carboxylic acid was fed to the bioreactorat 25 hours, to a final concentration of 5 mM. The fermentation brothwas harvested after 7 days and clarified by centrifugation to providesupernatant and cells. The cells (1 L) were extracted by mixingthoroughly with an equal volume of methanol then allowed to stand for 30minutes. The cell-methanol slurry was centrifuged, and the supernatantdecanted off. The procedure was repeated. The fermentation supernatant(12 L) was adjusted to pH˜10 by addition of 5 N NaOH and stirred gentlywith 0.75 volumes of ethyl acetate for 8 hours. The upper phase wasremoved by aspiration and the extraction repeated. The ethyl acetate andmethanol fractions were combined and the solvents removed in vacuo toyield a yellow-brown oil/aqueous mixture (1 L). This was mixed withethyl acetate (2 L) and extracted with a solution of 50 mM tartaric acid(3×1.5 L). The tartaric acid extracts were combined, adjusted to pH˜10with 5 N NaOH, and extracted with ethyl acetate (3×1.5 L). The ethylacetate extracts were combined and the solvent removed in vacuo to leavea brown oily residue (7.5 g). The residue was dissolved into ethylacetate (500 mL) and extracted three times with 50 mM tartaric acid (350mL). The tartaric acid fractions were combined, adjusted to pH˜10 andre-extracted with ethyl acetate (3×500 mL). The ethyl acetate fractionswere combined and the solvent removed in vacuo to yield a brown oilyresidue (0.5 g).

The oily residue was dissolved into methanol (1.5 mL) andchromatographed, in two equal portions, over base-deactivatedreversed-phase silica gel (Hypersil C₁₈-BDS, 5 μm; 21×250 mm) elutingwith a mobile phase gradient as described below, at a flow rate of 21mL/minute.

-   -   Mobile phase gradient: T=0 minute, 15% B; T=5, 35% B; T=35, 90%        B; T=45, 95% B.    -   Mobile-phase A: 10% acetonitrile/90% water, containing 10 mM        ammonium acetate and 0.15% formic acid.    -   Mobile-phase B: 90% acetonitrile/10% water, containing 10 mM        ammonium acetate and 0.15% formic acid.

Fractions were collected every 30 seconds. Fractions from the initialfractionation that contained 21-desethyl-21-cyclobutyl spinosyn A werecombined, and the solvent removed in vacuo. The residues werechromatographed over reversed-phase silica gel (Prodigy C₁₈, 5 μm;10×250 mm) eluting with a gradient as described below, at a flow rate of5 mL/minute.

T=0, 55% B; T=5, 70% B; T=35, 95% B; T=45, 95% B.

Fractions were collected every 30 seconds. Fractions containing the21-desethyl-21-cyclobutyl spinosyn A were combined, the acetonitrileremoved in vacuo, and the sample concentrated using C₁₈-BondElutecartridges (200 mg). The sample was applied under gravity, washed withwater (10 mL) and eluted with methanol (2×10 mL), then the solventremoved in vacuo. Fractions from the initial crude fractionation thatcontained 21-desethyl-21-cyclobutyl spinosyn D were combined and thesolvent removed in vacuo. The residues were chromatographed overreversed-phase silica gel (Prodigy C₁₈, 5 μm; 10×250 mm) eluting with agradient as described below, at a flow rate of 5 mL/minute.

-   -   T=0 minute, 25% B; T=5, 55% B; T=35, 95% B; T=45, 95% B.

Fractions were collected every 30 seconds. Fractions containing the21-desethyl-21-cyclobutyl spinosyn D were combined, the acetonitrileremoved in vacuo, and the sample concentrated using C₁₈-BondElutecartridges (200 mg capacity). The sample was applied under gravity,washed with water (10 mL) and eluted with methanol (2×10 mL), and thesolvent removed in vacuo.

The chemical structures of the new spinosyns were determined byspectroscopic methods, including nuclear magnetic resonance spectroscopy(NMR), mass spectrometry (MS), ultraviolet spectrometry (UV), coupledhigh performance liquid chromatography-mass spectrometry (HPLC-MS), andby comparison to the spectral data for the known compounds spinosyns A,D, E and F.

21-desethyl-21-cyclobutyl spinosyn A (Compound 3) has the followingcharacteristics:

-   -   Isolated yield: 3.1 mg    -   Molecular weight: 757    -   Molecular formula: C₄₃H₆₇NO₁₀    -   UV (by diode array detection during HPLC-MS analysis): 245 nm    -   Electrospray MS: m/z for [M+H]⁺=758.5; forosamine sugar fragment        ion at m/z=142.2.

Accurate FT-ICR-MS: m/z for [M+H]⁺=758.4830 (requires: 758.4838).

Table 6 shows the ¹H and ¹³C NMR chemical shift data for21-desethyl-21-cyclobutyl spinosyn A in CDCl₃.

TABLE 6 Position ¹H ¹³C  1 — 172.8   2a 2.43 33.8  2b 3.12 33.8  3 3.0147.5  4 3.54 41.7  5 5.80 128.8   6 5.88 129.3   7 2.17 41.1  8a 1.3736.2  8b 1.92 —  9 4.31 76.0 10a 1.33 37.3 10b 2.26 — 11 0.91 46.0 122.88 49.4 13 6.76 147.5  14 — 144.2  15 — 202.9  16 3.26 47.8 16-Me 1.1716.1 17 3.61 80.6 18a 1.49 34.4 18b 1.49 — 19a — 21.7 19b — — 20a 1.3428.2 20b 1.44 — 21 4.80 22 2.34 23a 1.65 23b 1.65 24a 1.76 17.8 24b 1.76— 25a 1.86 24.6 25b 1.86 —  1′ 4.85 95.4  2′ 3.49 77.7  3′ 3.46 81.0  4′3.12 82.2  5′ 3.54 67.9  6′ 1.28 17.8  2′-OMe 3.49 59.0  3′-OMe 3.5057.7  4′-OMe 3.56 60.9  1″ 4.42 103.4   2″a 1.47 30.8  2″b 1.98 —  3″a1.47 18.5  3″b 1.98 —  4″ 2.26 64.8  5″ 3.84 73.5  6″ 1.26 19.0  4″-NMe₂2.26 40.6 Chemical shifts referenced to the proton of CHCl₃ at 7.26 ppm

21-desethyl-21-cyclobutyl spinosyn D (Compound 4) had the followingcharacteristics:

-   -   Isolated yield: ˜1 mg    -   Molecular weight: 771    -   Molecular formula: C₄₄H₆₉NO₁₀    -   UV (by diode array detection during HPLC-MS analysis): 245 nm    -   Electrospray MS: m/z for [M+H]⁺=772.5; forosamine sugar fragment        ion at m/z=142.2.

The small quantity of material precluded detailed NMR study of thismolecule, but the data accumulated was consistent with the expectedstructure. This analysis was aided by comparison to the data for21-desethyl-21-cyclobutyl spinosyn A.

EXAMPLE 9 Preparation of 5,6-dihydro-21-desethyl-21-cyclobutyl spinosynA (Compound 8) and 5,6-dihydro-21-desethyl-21-n-propyl spinosyn A(Compound 29)

A solution of 21-desethyl-21-cyclobutyl spinosyn A (3.1 mg, 0.004 mmol)in 2 mL of toluene and 0.5 mL of ethanol was purged with a slow streamof nitrogen for 20 minutes, then 2 mg of chlorotris(triphenylphosphine)rhodium was added and the solution hydrogenated at 60° C. and 1 atm. for16 hours. After cooling and removal of solvent, the residue waschromatographed using a 10 cm×2 cm silica gel column, eluting with 5×25mL fractions of dichloromethane containing 0%, 2%, 3%, 4%, and 5% MeOHrespectively. The product-containing fractions were combined andconcentrated to give 2.1 mg of 5,6-dihydro-2′-desethyl-21-cyclobutylspinosyn A. MS M+ 760.

5,6-dihydro-21-desethyl-21-n-propyl spinosyn A (Compound 29) wasprepared from 21-desethyl-21-n-propyl spinosyn A (Compound 23) using thesame procedure.

EXAMPLE 10 Production and isolation of 21-desethyl-21-cyclopropylspinosyns A and D (Compounds 1 and 2)

Frozen vegetative stocks of S. spinosa 13E were inoculated into primaryvegetative pre-cultures in CSM (50 mL incubated in a 250 mL Erlenmeyerflask with spring). Secondary pre-cultures in vegetative medium (250 mLincubated in a 2 L Erlenmeyer flask with spring) were prepared andincubated as described in Example 8.

Fourteen liters of production medium were prepared, as in Example 6,with the addition of 0.01% v/v Pluronic L-0101 (BASF) antifoam.Production medium was inoculated with the secondary pre-culture at 5%v/v and allowed to ferment in a 20 L stirred bioreactor for 7-10 daysunder the conditions described in Example 8. Cyclopropyl carboxylic acidwas fed to the bioreactor at 45 hours to a final concentration of 5 mM.The fermentation broth was harvested after 7 days and extracted asdescribed in Example 8.

The oily residue was dissolved in methanol (1.5 mL) and initiallycrudely chromatographed as described in Example 8. Fractions from theinitial separation that contained 21-desethyl-21-cyclopropyl spinosyn Awere combined and the solvent removed in vacuo. The residues werechromatographed over reversed-phase silica gel (Prodigy C₁₈, 5 μM;10×250 mm) eluting with a gradient as described below, at a flow rate of5 mL/minute.

-   -   T=0 minute, 55% B; T=70% B; T=30, 95% B; T=35, 95% B.

Fractions were collected every 30 seconds. Fractions containing the21-desethyl-21-cyclopropyl spinosyn A were combined, the acetonitrileremoved in vacuo, and the sample concentrated using a C₁₈-BondElutecartridge (200 mg). The sample was applied under gravity, washed withwater (10 mL) and eluted with methanol (2×10 mL), and then the solventwas removed in vacuo.

21-desethyl-21-cyclopropyl spinosyn A (Compound 1) has the followingcharacteristics:

-   -   Isolated yield: ˜1 mg    -   Molecular weight: 743    -   Molecular formula: C₄₂H₆₅NO₁₀    -   UV (by diode array detection during HPLC-MS analysis): 245 nm    -   Electrospray MS: m/z for [M+H]⁺=744.5; forosamine sugar fragment        ion at m/z=142.2.

Table 7 summarizes the ¹H and ¹³C NMR spectral data for21-desethyl-21-cyclopropyl spinosyn A in CDCl₃.

TABLE 7 Position ¹H ¹³C  1 — —  2a 2.43 34.0  2b 3.13 —  3 3.00 47.3  43.49 41.4  5 5.87 129.1   6 5.79 128.6   7 2.15 —  8a 1.34 36.0  8b 1.91—  9 4.30 75.9 10a 1.32 37.0 10b 2.24 — 11 0.89 45.9 12 2.85 34.0 136.74 147.4  14 — — 15 — — 16 3.26 47.5 17 3.62 80.6 18a 1.51 34.2 18b1.51 — 19a 1.18 22.0 19b 1.74 — 20a 1.62 30.9 20b 1.62 — 21 4.18 79.3 220.89 16.5 23a* 0.44  2.3 23b* 0.14 — 24a* 0.44  3.7 24b* 0.14 — 25 1.1716.0  1′ 4.84 95.2  2′ 3.49 77.5  3′ 3.45 80.9  4′ 3.10 82.1  5′ 3.5467.7  6′ 1.27 17.6  2′-OMe 3.49 58.8  3′-OMe 3.48 57.5  4′-OMe 3.55 60.7 1″ 4.41 103.4   2″a 1.47 30.8  2″b 1.98 —  3″a 1.45 18.2  3″b 2.24 — 4″ 2.24 64.7  5″ 3.48 73.4  6″ 1.27 18.7  4″-NMe₂ 2.24 40.6 Chemicalshifts referenced to the proton of CHCl₃ at 7.26 ppm. *These assignmentsare interchangeable.

21-desethyl-21-cyclopropyl spinosyn D (Compound 2) has the followingcharacteristics:

-   -   Isolated yield: ˜0.5 mg    -   Molecular weight: 757    -   Molecular formula: C₄₃H₆₇NO₁₀    -   UV (by diode array detection during HPLC-MS analysis): 245 nm

Electrospray MS: m/z for [M+H]⁺=758.5; forosamine sugar fragment ion atm/z=142.2.

The small quantity of material precluded the detailed NMR study of thismolecule, but the data accumulated was consistent with the expectedstructure. This analysis was aided by comparison to the data for21-desethyl-21-cyclopropyl spinosyn A.

b. Hybrid Spinosyn PKS Using Ave Loading Domain

The loading module of the avermectin biosynthetic cluster (aveAT0ACP0)governs the introduction of C-2 branched starter units into theavermectin molecule, derived from iso-butyryl-CoA and2-methylbutyryl-CoA. There is precedent for the swapping of this loadingdomain into the erythromycin PKS pathway to give novel polyketides withthe starter unit specificity of the avermectin system (WO 98/01546, WO98/01571, Marsden et al., 1998). The avermectin loading module has alsobeen shown to incorporate CoA-esters of a broad range of free acids fromthe production medium, either in its native environment, or as part ofthe ave/ery hybrid pathway (Pacey et al., 1998). The avermectin loadingmodule swap described in the literature is actually a replacement of theerythromycin AT0ACP0 by the avermectin AT0ACP0. This leads to a piece oferythromycin DNA sequence upstream of the avermectin AT0 between thestart codon and a SpeI site. This was included in the ave/ery experimentbecause the N-terminal region (upstream of the homologous AT domain) ismuch larger in the erythromycin loading module than in that ofavermectin, and may be important for stability of the protein. Becausethe resulting hybrid had been productive, the same region was used forthe ave/spn hybrid. In effect, the resulting hybrid gene is anery/ave/spn hybrid, but since it transfers the specificity of theavermectin loading module to the spinosyn PKS, it has been designated anave/spn hybrid through-out.

The avermectin loading module (AT0ACP0) was cloned from pIG1 (WO98/01546, WO 98/01571, Marsden et al., 1998) in-frame and upstream ofthe spnKS1, under the control of either P_(actI), or P_(ptr). The samesplice site, at the beginning edge of the KS1 homologous region, wasused as for the ery load experiment described above. A region ofhomology for integration was incorporated from pRHB3E11. The resultingplasmids, pLSB29 (with the hybrid PKS region under the control ofP_(actI)) and pLSB30 (with the hybrid PKS region under the control ofP_(ptr)) are based on pKC1132 and, therefore, contain the apramycinresistance marker for selection both in E. coli and S. spinosa, and theoriT for conjugal transfer of DNA from E. coli to S. spinosa (Bierman etal., 1992, Matsushima et al., 1994). These constructs were transformedinto S. spinosa NRRL 18538 by conjugation. Exconjugants were confirmedto contain the hybrid ave/spn PKS gene under the appropriate promoter byPCR analysis.

S. spinosa NRRL 18538:pLSB29 was designated S. spinosa 21K2. It producedmainly spinosyns E, A and D by incorporation of acetate and propionateinto the loading module. Additional small peaks were observed in LC-MS,with masses that were consistent with the novel natural products21-desethyl-21-iso-propyl spinosyn A and 21-desethyl-21-sec-butylspinosyn A and the equivalent D analogues. These minor products resultedfrom incorporation of iso-butyrate and 2-methyl butyrate respectivelyinto the starter. In avermectin biosynthesis itself, the ave loadingmodule recruits only these branched starters. However, a broaderspectrum of products was observed when the ave loading module wasspliced upstream of the KS1 in the erythromycin PKS, indicating thatsuch a hybrid can incorporate acetate and propionate as well. Wetherefore observed the expected range of products from this engineeredS. spinosa strain.

The structure of isolated 21-desethyl-21-iso-propyl spinosyn A wasconfirmed by NMR characterization. 21-desethyl-21-n-propyl spinosyn Awas isolated as a minor component from the production culture of21-desethyl-21-iso-propyl spinosyn A. The 21-n-propyl analogue,presumably made by the loading module taking butyrate from the medium,was also fully characterized. Both were active in insect control assays.

Some of the natural versatility of the loading module of the avermectinPKS had previously been transferred to the erythromycin system, so abroad range of free acids was fed to S. spinosa 21K2 to generatespinosyns with altered starter units. Novel spinosyn compounds wereidentified on the basis of UV chromophore, mass and mass spectralfragmentation, along with the knowledge of which acid was fed and,therefore, which compounds were expected from each experiment. Toconfirm the structure predictions made, a number of spinosyn analogueswith novel C21 starter groups were isolated and fully characterized.

Free acids that can be used in this way include, but are not limited to,the following: Cyclic organic acids including cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cyclohexenyl and 2-methylcyclopropyl;heteroatom containing cyclic organic acids including 2-furoic acid,3-furoic acid, thiophene carboxylic acid and methyl thiophene carboxylicacid; branched chain carboxylic acids; and some other acids, whichincluded methylthioacetic acid, chloroacetic acid, cyanoacetic acid andmethoxyacetic acid.

Such feeding experiments lead to analogues of spinosyns (a spinosynbeing a macrolide with a 22-membered tetracyclic nucleus of the typeillustrated in Tables 1 and 2 and in Formula I) in which C21 bears aside chain other than ethyl, generally an alpha-branched C2-C5 alkylgroup, a C3-C8 cycloalkyl or cycloalkenyl group (optionally substituted,e.g., with one or more hydroxy, C1-C4 alkyl or alkoxy groups or halogenatoms), or a 3-6 membered heterocycle containing O or S, saturated orfully or partially unsaturated, optionally substituted (as forcycloalkyl). Preferred candidates for the C21 substituent are the groupsof carboxylate units useable as substrates by the ery or ave loadingmodules. Preferred substrates include cyclobutane carboxylic acid andcyclopropane carboxylic acid. Other possibilities include n-butyricacid, iso-propyl carboxylic acid, 2-(S)-methylbutyric acid,2-methylcyclopropane carboxylic acid, 3-furoic acid and methylthioaceticacid.

Feeding the branched chain acids iso-propyl and 2-methyl butylcarboxylic acid did not alter the yield of novel natural products madedue to the normal specificity of the ave load. These products wereobserved in all healthy cultures of S. spinosa 21K2 in low yield.

Cyclobutyl carboxylic acid was accepted by the ave loading module togive the A and D analogues, and a substantial proportion of theC17-pseudoaglycone of the A analogue. The cyclobutyl products wereobserved at higher levels than in the fed hybrid ery/spn system, with21-desethyl-21-cyclobutyl spinosyn A at ˜5-10 mg/liter,21-desethyl-21-cyclobutyl spinosyn D at lower but significant levels and21-desethyl-2′-cyclobutyl spinosyn A 17-pseudoaglycone at ˜10-15mg/liter. The cyclopropyl carboxylic acid feed was also successful,yielding 21-desethyl-21-cyclopropyl spinosyns A and D. The twocyclobutyl analogues and the cyclopropyl A analogue were shown to beidentical to the purified compounds isolated from the ery loadexperiment.

Methylthioacetic acid was incorporated to give the21-desethyl-21-methylthiomethyl spinosyn A, at yields comparable tothose of the cyclobutyl experiment above. This was a significantimprovement in yield over production of the same compound from the eryload experiment. 2-furoic acid and methylcyclopropyl carboxylic acidwere also incorporated to give the expected novel products.

EXAMPLE 11 Construction of a Vector to Incorporate the Loading Module ofthe Avermectin Polyketide Synthase into the Spinosyn Polyketide Synthase

See FIG. 5. The vector to incorporate the loading module of theavermectin polyketide synthase into the spinosyn polyketide synthasecontains the avermectin loading module (AT0ACP0) followed by a region ofthe first module of the spinosyn PKS to provide homology forintegration. The vector is designated pLSB29 and was constructed asfollows:

The avermectin loading module has been previously spliced upstream ofthe erythromycin module 1 (WO 98/01546, WO 98/01571, Marsden et al.,1998). In this ave/ery hybrid the loading module had the DNA coding forthe ery amino acid sequence at the start, followed by the AT0ACP0 of theave PKS. This hybrid fragment conferred the specificity of theavermectin loading domain, although it included a small amount of erysequence. The same fragment was used in this experiment, and leads to anery/ave/spn hybrid protein. We describe it simply as an ave/spn hybridsince it is the specificity of the avermectin loading domain that isconferred on the spinosyn pathway. The fragment begins with an NdeI siteincorporating the start codon, and ends with a NheI site engineered atthe beginning of the KS1. This introduces a conservative amino acidchange (Ile-Leu) in the spinosyn KS1 sequence.

To introduce the NheI site, the region of spnA from the beginning of theKS1 was amplified by PCR using pRHB3E11 (U.S. Pat. No. 6,274,350 B1,Waldron et al., 2001) as the template, and oligos SP14 (SEQ ID NO:9) andSP15 (SEQ ID NO:10). SP14 introduces an NheI site at bases 24107-24112(numbers refer to SEQ ID NO:1 of U.S. Pat. No. 6,274,350). SP15 bindsapproximately 1500 bp downstream, at a BstEII site (25646-25652). A NotIsite was also incorporated into SP15 for the subsequent cloning step.The PCR was carried out using Pwo thermostable polymerase under standardconditions.

The PCR product was phosphorylated, gel-purified and cloned into pUC19,which had been previously digested with SmaI and dephosphorylated. Anumber of insert-containing clones were sequenced. One clone containingthe insert in the orientation that places the NheI site next to theEcoRI site in the vector was designated pLSB5.

To provide a large enough region of homology for integration, a fragmentof approximately 2.6 kbp (from BstEII to NotI) was cloned out ofpRHB3E11 into pLSB5, to yield pLSB8.

The avermectin loading module was then cloned from pIG1 (WO 98/01546, WO98/01571, Marsden et al., 1998) as an NdeI/NheI fragment and ligatedinto pLSB8 previously digested with NdeI and NheI. The DNA sequence ofthe avermectin loading module fragment used, from the NdeI site to theNheI site is shown in SEQ ID NO:11. The resulting plasmid was designatedpLSB14.

The fragment contained in pLSB14 contains the avermectin loading modulespliced in-frame to the spinosyn KS1, with a region of homology to spnA,which is sufficient for integration to occur.

This fragment was then removed as an NdeI/XbaI fragment and cloned intopLSB2 to give pLSB29. This places the new ave/spn hybrid fragment underPactI, in a vector that can be transferred into S. spinosa byconjugation and then selected using the apramycin resistance marker.

EXAMPLE 12 Generation of a S. Spinosa Strain Harboring a HybridPolyketide Synthase Comprising the Avermectin Loading Module Fused tothe KS1 of the Spinosyn PKS

See FIG. 6. Saccharopolyspora spinosa NRRL 18538 was transformed byconjugation (Matsushima et al., 1994) from E. coli S17-1 (Simon et al.,1983) with pLSB29. Transformants were selected by resistance toapramycin and screened by Southern blot analysis. A single transformantdisplaying the correct hybridization pattern to show that the plasmidhad integrated into the chromosome by homologous recombination wasdesignated strain S. spinosa 21K2.

EXAMPLE 13 Production of Metabolites by S. spinosa 21K2 Fermentation(Production of Compounds 9-12)

S. spinosa 21K2 was cultured from a frozen vegetative stock used toinoculate CSM medium (Hosted and Baltz 1996; U.S. Pat. No. 5,362,634).This pre-culture was grown in flasks shaken at 300 rpm with a one-inchthrow at a temperature of 30° C. for 3 days. This was used to inoculatevegetative medium (Strobel and Nakatsukasa 1993; U.S. Pat. No.5,362,634) at 5% v/v and was cultured under the same conditions for afurther 2 days. The vegetative culture was used to inoculate productionmedium (Strobel and Nakatsukasa 1993) at 5% v/v. Small-scale productioncultures were fermented under the same conditions as the pre-cultures,but for 7-10 days at 250 rpm with a two-inch throw and at 75% relativehumidity. Initial small-scale production cultures were grown in 6 mL ofproduction medium in 25 mL Erlenmeyer flasks with springs for 7 days.

To identify the metabolites produced, a 1 mL aliquot of fermentationbroth was analyzed by LC-MS as described in Example 5. By comparison toauthentic standards, and to a fermentation extract from strain S.spinosa NRRL 18538, the major compounds produced by S. spinosa 21K2 werespinosyns A, D and E. Spinosyn A was the major component produced, andthe total yield of spinosyns was ˜50 mg/l.

In addition to the known spinosyns A, D and E, four new compounds wereclearly present. The chromatographic retention time and mass spectraldata for these new compounds (Table 8) were consistent with theirsynthesis through the incorporation of branched chain starter units(iso-propyl carboxylic acid and 2-methylbutyric acid). The MS spectra ofthe new compounds gave ions for the [M+H]+ species and for theforosamine fragment (142.3). The compounds derived from an iso-propylcarboxylic acid feed were present 2-3× higher levels than those derivedfrom 2-methylbutyric acid. The new compounds comprised no more than 5%of the total spinosyns present.

TABLE 8 Compound Retention Carboxylic acid starter No. (see time Keymass spectral unit Table 3) (min) data (m/z) iso-propyl carboxylic 925.1 746.5 [M + H]+; 142.3 acid iso-propyl carboxylic 10 26.3 760.5 [M +H]+; 142.3 acid 2-methylbutyric acid 11 26.7 760.4 [M + H]+; 142.32-methylbutyric acid 12 27.5 774.5 [M + H]+; 142.3

EXAMPLE 14 Precursor-Directed Production of Novel Spinosyns from S.Spinosa 21K2 (Production of Compounds 1-6 and 13-20)

The ave/spn hybrid PKS was used to generate novel spinosyn metabolitesby feeding carboxylic acids to production cultures. The avermectinloading module incorporated the carboxylic acid within the starter ofthe molecule.

Parallel 6 mL production flasks were inoculated as described in Example13 above. After 24 hours, each of these was fed with a carboxylic acid(stock solutions made in water and pH adjusted to 6.5 with sodiumhydroxide) at a final concentration of 3 mM. After 7 days a 1 mL aliquotof fermentation broth was analyzed by LC-MS as described in Examples 6and 12. The incorporation of cyclobutyl carboxylic acid, cyclopropylcarboxylic acid, 2-methylcyclopropyl carboxylic acid, methylthio aceticacid and 3-furoic acid provided novel C21-modified spinosyns, asindicated by the appearance of new peaks in the UV and MS chromatograms(Table 9). The mass spectra of the novel compounds gave ions for the[M+H]⁺ species and for the forosamine fragment (142.3). In addition thefeeding of cyclobutyl- and cyclopropyl carboxylic acids also causedsignificant accumulation of the corresponding 17-pseudoagylcones.

TABLE 9 Compound Retention No. (see time Key mass spectral Carboxylicacid fed Table 3) (minute) data (m/z) cyclobutyl CA 2 25.7 758.4 [M +H]⁺; 142.4 cyclobutyl CA 3 27.2 772.5 [M + H]⁺; 142.4 cyclopropyl CA 123.5 744.5 [M + H]⁺; 142.4 cyclopropyl CA 2 25.1 758.5 [M + H]⁺; 142.42-methyl cyclopropyl 13 25.7 758.5 [M + H]⁺; 142.4 CA 2-methylcyclopropyl 14 26.9 772.5 [M + H]⁺; 142.4 CA methylthio acetic acid 522.9 764.5 [M + H]⁺; 142.4 methylthio acetic acid 6 24.4 778.4 [M + H]⁺;142.4 3-furoic acid 15 22.9 770.5 [M + H]⁺; 142.4 3-furoic acid 16 24.3784.5 [M + H]⁺; 142.4 cyclobutyl CA 19 27.1 639.4 [M + Na]⁺ cyclobutylCA 20 28.6 653.4 [M + Na]⁺

The 21-cyclobutyl and cyclopropyl compounds were confirmed as thecorrect structures in comparison to the compounds isolated from feedingthe ery load strain, S. spinosa 13E.

EXAMPLE 15 Isolation of Novel Metabolites from a Large-ScaleFermentation of S. Spinosa 21K2

Frozen vegetative stocks of S. spinosa 21K2 were inoculated into primaryvegetative pre-cultures of S. spinosa 21K2 in CSM (50 mL incubated in a250 mL Erlenmeyer flask with spring). Secondary pre-cultures invegetative medium (250 mL incubated in a 2 L Erlenmeyer flask withspring) were prepared and incubated as described in Example 6.

Fourteen liters of production medium were prepared, as in Example 6,with the addition of 0.01% v/v Pluronic L-0101 (BASF) antifoam.Production medium was inoculated with the secondary pre-culture at 5%v/v and allowed to ferment in a 20 L stirred bioreactor for 7-10 daysunder the conditions described in Example 8. 2-Methyl butyric acid, to afinal concentration of 2 mM, was fed to the bioreactor at 26 and 37.5hours, leading to an overall final concentration of 4 mM. Thefermentation broth was harvested after 7 days and extracted as describedin Example 8.

The oily residue was dissolved in methanol (1.5 mL) and initiallychromatographed as described in Example 8. Fractions from the initialseparation that contained 21-desethyl-21-iso-propyl spinosyn A werecombined and the solvent removed in vacuo. The residues werechromatographed over reversed-phase silica gel (Prodigy C₁₈, 5 μm;10×250 mm) eluting with a gradient as described below, at a flow rate of5 mL/minute.

-   -   T=0, 55% B; T=5, 70% B; T=35, 95% B; T=45, 95% B.

Fractions were collected every 30 seconds. Fractions containing the21-desethyl-21-iso-propyl spinosyn A were combined, the acetonitrileremoved in vacuo, and the sample concentrated using C₁₈-BondElutecartridges (200 mg). The sample was applied under gravity, washed withwater (10 mL) and eluted with methanol (2×10 mL), then the solvent wasremoved in vacuo.

The dried sample was dissolved in methanol (0.5 mL) and chromatographedover reversed-phase base-deactivated silica gel (hypersil-C18-BDS;4.6×250 mm; 5 μm). The column was eluted isocratically at a flow rate of1 mL/minute with ammonium acetate (10 mM)-methanol-tetrahydrofuran(40:45:15), and the three major components were collected after UVdetection (244 nm). Under these conditions, retention times were asfollows: spinosyn-D: 64.4 minutes, 21-desethyl-21-iso-propyl spinosyn A:67.8 minutes and 21-desethyl-21-n-propyl spinosyn A: 75.3 minutes. Eachsample was dried in vacuo to give a white solid, which was identified byits NMR and mass spectra.

21-desethyl-21-n-propyl spinosyn A (Compound 23) has the followingcharacteristics.

-   -   Molecular Weight: 745    -   Molecular formula: C₄₂H₆₇NO₁₀    -   UV (by diode array detection during HPLC analysis): 244 nm    -   Electrospray MS: m/z for [M+H]+=746.5; forosamine sugar fragment        ion at m/z=142.2.

Table 10 summarizes the ¹H and ¹³C NMR chemical shift data for21-desethyl-21-n-propyl spinosyn A in CDCl₃.

TABLE 10 Position ¹H ¹³C  1 — 172.5   2a 3.13 34.1  2b 2.42 —  3 3.0347.5  4 3.50 41.5  5 5.81 128.8   6 5.89 129.3   7 2.19 41.1  8a 1.9436.2  8b 1.35 —  9 4.32 76.0 10a 2.29 37.3 10b 1.35 — 11 0.92 45.9 122.88 49.4 13 6.78 144.1  14 — 147.5  15 — 202.9  16 3.30 47.6 16-Me 1.2016.2 17 3.65 80.6 18 1.55 34.3 18b 1.55 — 19a 1.78 21.6 19b 1.21 — 20a1.56 30.7 20b 1.50 — 21 4.77 75.3 22a 1.50 37.8 22b 1.39 — 23 1.28 18.324 0.99 14.0  1′ 4.88 95.4  2′ 3.51 77.7  3′ 3.49 81.0  4′ 3.13 82.2  5′3.56 67.9  6′ 1.30 19.0  2′-OMe 3.51 59.0  3′-OMe 3.51 57.7  4′-OMe 3.5760.9  1″ 4.43 103.5   2″a 1.99 30.9  2″b 1.49 —  3″a 1.38 18.5  3″b 1.50—  4″ 2.25 64.8  5″ 3.49 73.5  6″ 1.28 17.8  4″-NMe₂ 2.26 40.6 Chemicalshifts referenced to the proton of CHCl₃ at 7.26 ppm

21-desethyl-21-iso-propyl spinosyn A (Compound 9) has the followingcharacteristics.

-   -   Molecular Weight: 745    -   Molecular formula: C₄₂H₆₇NO₁₀    -   UV (by diode array detection during HPLC analysis): 244 nm    -   Electrospray MS: m/z for [M+H]+=746.5; forosamine sugar fragment        ion at m/z=142.2.

Table 11 summarizes the ¹H and ¹³C NMR chemical shift data for21-desethyl-21-iso-propyl spinosyn A in CDCl₃.

TABLE 11 Position ¹H ¹³C  1 — 173   2a 3.15 34  2b 2.43 —  3 3.03 48  43.55 42  5 5.82 129   6 5.90 129   7 2.19 42  8a 1.94 36  8b 1.35 —  94.32 76 10a 2.28 37 10b 1.38 11 0.93 46 12 2.90 50 13 6.78 144  14 —147  15 — 204  16 3.28 48 16-Me 1.19 16 17 3.63 81 18 1.52 35 18b 1.52 —19a 1.80 22 19b 1.19 — 20a 1.52 27 20b 1.52 — 21 4.66 80 22 1.80 33 230.85 18 22-Me 0.83 18  1′ 4.87 96  2′ 3.51 78  3′ 3.47 81  4′ 3.13 82 5′ 3.56 68  6′ 1.30 19  2′-OMe 3.51 59  3′-OMe 3.51 58  4′-OMe 3.58 61 1″ 4.43 104   2″a 1.99 31  2″b 1.49 —  3″a 1.38 19  3″b 1.50 —  4″ 2.2565  5″ 3.49 74  6″ 1.28 18  4″-NMe₂ 2.26 41 Chemical shifts referencedto the proton of CHCl₃ at 7.26 ppm; 13C data from 2D experiments.

Fractions from the initial fractionation that contained the putative21-desmethyl-21-sec-butyl spinosyn A were combined, the acetonitrileremoved in vacuo, and concentrated using C₁₈-BondElute cartridges (200mg). The sample was applied under gravity, washed with water (10 mL) andeluted with methanol (2×10 mL), then the solvent removed in vacuo.

The putative 21-desmethyl-21-sec-butyl spinosyn A (Compound 11) has thefollowing characteristics.

-   -   Molecular Weight: 759    -   Molecular formula: C₄₃H₆₉NO₁₀    -   UV (by diode array detection during HPLC analysis): 244 nm    -   Electrospray MS: m/z for [M+H]+=760.5; forosamine sugar fragment        ion at m/z=142.2.        Coexpression of S. Cinnamonensis Crotonyl-CoA Reductase\

In another aspect, the invention provides engineered S. spinosa hoststhat present an altered substrate supply such that the native polyketidesynthase produces novel products. For example, co-expression of the S.cinnamonensis crotonyl-CoA reductase with the spinosyn biosyntheticpathway leads to novel products where the loading module additionallyincorporates ethyl malonyl-CoA to yield 21-desethyl-21-n-propylspinosyns A and D, and the AT of module 8 additionally incorporatesethyl malonyl-CoA to yield 6-ethyl spinosyn A. In addition, both ofthese AT domains can accept ethyl malonyl-CoA to yield6-ethyl-21-desethyl-21-n-propyl spinosyn.

The spinosyn loading module comprises a KSqAT0ACP0, which predominantlydecarboxylates methyl malonyl-CoA to incorporate propionate. Loadingmodules containing a KSq are generally more specific than those thatlack them. It is, therefore, interesting and unusual that the spinosynloading module occasionally accepts malonyl-CoA (naturally producingtrace amounts of spinosyn E) and, as disclosed here, unexpectedlyaccepts ethyl malonyl-CoA when it is present.

For evidence supporting this concept see Example 18 and the discussionimmediately preceding Example 22.

Hybrid Spinosyn PKS with Heterologous Extension Modules

In another of its aspects the invention provides hybrid spinosyn PKSscomprising a heterologous extension module or a spinosyn extensionmodule with a heterologous AT domain.

The AT domains select the extender units that are incorporated into thegrowing polyketide chain. In spinosyn biosynthesis, the extender ispredominantly malonyl-CoA. However, the AT in module 3 is essentiallyspecific for methyl malonyl-CoA. It occasionally incorporatesmalonyl-CoA, leading to the accumulation of spinosyn F as a naturalminor component. The AT in module 8 shows a relaxed specificity,incorporating predominantly malonyl-CoA (to give spinosyn A) but alsoincorporating a significant amount methyl malonyl-CoA (to give about 15%spinosyn D). The amino acid sequence of the module 8 AT is similar toother AT domains in regions that are associated with the selection ofmethyl malonyl-CoA. We suggest, therefore, that the incorporation ofpredominantly malonyl-CoA at this position is a reflection of substratesupply, in combination with an AT domain that has an unusually loosespecificity. The AT of the loading module is largely selective formethyl malonyl-CoA, but incorporates malonyl-CoA 5-10% of the time(leading to production of the natural minor component spinosyn E).

Replacement of an existing AT domain with a heterologous AT domainselective for a different malonyl-CoA leads to a PKS that will add,remove or replace a side chain on the spinosyn polyketide. Where theheterologous AT domain can incorporate an extender molecule that is notreadily available within the cell, accessory genes are included toprovide the co-substrate (Stassi et al., 1998). For Example, there is noobvious gene encoding a crotonyl-CoA reductase in the genome of S.spinosa (C. Waldron, unpublished observation) and, therefore, it isanticipated that the supply of ethyl malonyl-CoA is severely limited inthis organism.

A system was constructed to allow AT swaps to be carried out in module 3of the spinosyn polyketide synthase. The AT of module 3 naturallyincorporates methyl malonyl-CoA and introduces the 16-methyl branch inspinosyn. One way to effect an AT swap is by replacement, which willresult in a strain with the same transcripts as in the native PKS. Analternative method is via single integration, which places the completeplasmid sequence into the spinosyn PKS genes and requires that apromoter be introduced to drive the genes downstream of the integrationsite.

The following Examples (Examples 16-19) describe AT domain-swapexperiments that involve subcloning fragments using the enzyme MscI.MscI is affected by dcm methylation due to the sequences surrounding thesite. Plasmids that are required to be digested with MscI are,therefore, passaged through E. coli ET12567, a dcm⁻ strain, to generateDNA that is not methylated by dcm.

A. Hybrid Spinosyn PKS Using Module 2AT of the Rapamycin PKS in Place ofSpinosyn Module 3 AT

Using the single integration approach, a spinosyn PKS was generated inwhich the module 3 AT (specific for methyl malonyl-CoA) was replaced bythe rapamycin module 2 AT (malonyl-CoA specific). The resulting strainwas designated S. spinosa 7D23. The PKS genes of 7D23 contain spnA, spnBand a truncated spnC under the native spnA promoter, followed by theplasmid sequence (including the apramycin resistance marker) and thehybrid spnC and spnD and spnE under control of an introduced promoter.In 7D23 the heterologous promoter used was the promoter for resistanceto pristinamycin. Alternative promoters could also be used, for examplethe actI promoter with its cognate activator actII-ORF4.

S. spinosa 7D23 produced the four predicted spinosyn analogues, spinosynF, spinosyn F 17-pseudoaglycone, 16-desmethyl spinosyn D and16-desmethyl spinosyn D 17-pseudoaglycone. The yields were unexpectedlyhigh, being only 3-fold lower than from a non-engineered strain (seeTable 12 within Example 20). The spinosyn F compounds have beenidentified previously, as minor components in non-engineered strains,but this manipulation of the PKS genes resulted in a dramatic increasein their yield.

The abundance of the pseudoaglycone F may be a reflection of a reducedactivity of the forosamine glycosyl transferase for this novelsubstrate. The final glycosylation occurs at C-17, so the neighboringC-16 methyl group may be important for substrate recognition.

EXAMPLE 16 Construction of a Vector Containing Spinosyn Module 3 AT withFlanking Restriction Sites

See FIGS. 7 a and 7 b. The spinosyn module 3 AT domain was amplified byPCR using pRHB3E11 as the template and the primers CR322 (SEQ ID NO:12)and CR323 (SEQ ID NO:13). These primers introduce MscI and AvrII sites(bp 3-8 of SEQ ID NOS:12 and 13) flanking the AT domain. The 973 bp PCRproduct was phosphorylated and cloned into commercially available pUC18,which had previously been digested with SmaI and dephosphorylated.Insert-containing transformants were screened for orientation andsequenced. One transformant, carrying an insert of the correct sequencein the orientation that places the AvrII site next to the HindIII siteof the polylinker, was designated pALK6.

The flanking region downstream of the spinosyn module 3 AT was amplifiedby PCR using pRHB3E11 as the template and the primers CR324 (SEQ IDNO:14) and CR332 (SEQ ID NO:15). Primer CR324 introduces an AvrII site(bp 3-8 of SEQ ID NO:14) at exactly the same position as CR323, andCR332 binds downstream of a PstI site, which is naturally occurring inthe sequence. The 1557 bp PCR product was phosphorylated and cloned intocommercially available pUC18, which had previously been digested withSmaI and dephosphorylated. Insert-containing transformants were screenedfor orientation and sequenced. One transformant, carrying an insert ofthe correct sequence in the orientation that places the AvrII siteadjacent to the HindIII site of the polylinker, was designated pALK9.pALK9 was digested with AvrII and PstI. The 1525 bp fragment wasgel-purified and cloned into pALK6 digested with AvrII and PstI, to giveplasmid pALK11.

pALK12 was constructed to provide a suitable polylinker for thisexperiment. pALK6 was digested with NdeI and PstI and ligated with anannealed linker of oligos CR328 (SEQ ID NO:16) and CR329 (SEQ ID NO:17).The linker was designed to destroy the NdeI site and leave XbaI, EcoRVand PstI sites. Insert-containing clones were analyzed by restrictionenzyme digestion and a single clone displaying the correct pattern wasdesignated pALK12.

The flanking region upstream of the spinosyn module 3 AT was amplifiedby PCR using pRHB3E11 as the template and the primers CR330 (SEQ IDNO:18) and CR321 (SEQ ID NO:19). Primer CR330 introduces an NdeI site(bp 18-23 of SEQ ID NO:18) at the ATG start codon of the spnC gene, andCR321 incorporates an MscI site (bp 3-8 of SEQ ID NO:19) at exactly thesame position as CR322. The 1729 bp PCR product was phosphorylated andcloned into commercially available pUC18, which had previously beendigested with SmaI and dephosphorylated. Insert-containing transformantswere screened for orientation and sequenced. One transformant, carryingan insert of the correct sequence in the orientation that places theMscI site adjacent to the HindIII site of the polylinker, was designatedpALK23. pALK12 was digested with PstI and XmnI and the 601 bp clonedinto PstI/XmnI digested pALK23 to give pALK24. pALK11 was digested withPstI and MscI and the 2488 bp fragment was ligated into the 3777 bpbackbone produced by digesting pALK24 with PstI and MscI, to givepALK25. To ensure sufficient homology for integration, the fragment ofDNA from the PstI site (bp 39607-39612 of SEQ ID NO:1 in U.S. Pat. No.6,274,350 B1) to the MscI site (bp 41189-41194) was excised frompRHB3E11 and cloned into pALK25 previously digested with PstI and EcoRVto give pALK26.

pALK26 is the intermediate plasmid in the experiments to generatespinosyn PKS genes with AT swaps in module 3. It contained the spinosynAT3 with flanking restriction sites, the upstream region of spnC to thestart codon, and a region downstream for homologous recombination. Itwas missing a 407 bp MscI-MscI fragment just upstream of the MscI siteat the edge of the AT, which has to be inserted after the AT swap hasbeen achieved.

EXAMPLE 17 Construction of a Vector that can be Used to Engineer theSpinosyn Biosynthetic Pathway to Produce C-16 Desmethyl Spinosyns

See FIG. 8. Plasmid pALK39 was used to integrate into the chromosome ofS. spinosa by homologous recombination. It was designed to incorporatethe rapamycin AT2 into spinosyn module 3. Such an engineered S. spinosastrain produced C-16 desmethyl spinosyns and intermediates in thispathway. Plasmid pALK39 was constructed as described below.

The rapamycin module 2 was excised from pCJR26 as an MscI/AvrII fragment(see SEQ ID NO:20) and ligated into pALK26, which had previously beendigested with MscI and AvrII, to give pALK28. The 407 bp MscI fragmentmissing from this construct was then excised from pALK24 (Example 16)and ligated into dephosphorylated, MscI-digested pALK28. Clones werescreened for the orientation of the insert, and a single clonecontaining the insert in the correct orientation was designated pALK32.pALK32 contains the required fragment to introduce the rap AT2 swap intomodule 3 of S. spinosa, with an NdeI site at the start codon and adownstream XbaI site just outside of the polyketide synthase sequence.This region was excised as an NdeI/XbaI fragment and cloned into pLSB3to give pALK39. The new module 3 hybrid fragment was thereby placedunder P_(ptr), in a vector that can be transferred into S. spinosa byconjugation and selected by apramycin resistance.

EXAMPLE 18 Construction of a Vector Capable of Over-Expressing aCrotonyl-CoA Reductase

See FIG. 9. In order to address substrate supply issues, pALK21 wasconstructed to over-express the crotonyl-CoA reductase (ccr) gene fromStreptomyces cinnamonensis. pALK21 was constructed as described below.

The S. cinnamonensis ccr gene was amplified by PCR from genomic DNAisolated from S. cinnamonensis ATCC 15413 using primers CCRMONF (SEQ IDNO:21) and CCRMONR (SEQ ID NO:22). CCRMONF introduces an NdeI site (bp7-12 of SEQ ID NO:21) that incorporates the ATG of the start codon ofthe gene, and CCRMONR introduces a BamHI site (bp 6-11 of SEQ ID NO:22)downstream of the stop codon. Amplification to obtain the ccr gene wascarried out under standard conditions using Pwo thermostable DNApolymerase. The fragment was phosphorylated with T4 polynucleotidekinase then cloned into commercially available pUC18 digested with SmaIand dephosphorylated. Insert-containing plasmids were sequenced, and oneplasmid containing the correct sequence was designated pLSB46. PlasmidpLSB46 contains the S. cinnamonensis ccr gene in the orientation thatplaces the introduced BamHI site adjacent to the XbaI site of thepolylinker. SEQ ID NO:23 shows the ccr gene from the NdeI site at thestart codon to the BamHI site after the stop codon.

The ccr gene was excised as an NdeI/XbaI fragment and cloned into theexpression vector pLSB2 (Example 2) digested with NdeI and XbaI, to givepLSB59. Plasmid pLSB59 contains the ccr gene under control of the actIpromoter, and the activator actII-ORF4. In order to co-express the ccrgene with an engineered hybrid PKS, the ccr-containing fragment, alongwith the actII-ORF4 activator and actI promoter, was transferred into avector containing a second promoter (Pptr), which has restriction sitesavailable for a hybrid PKS gene. This was achieved as described below.

Plasmid pLSB59 was digested with NdeI, and the ends were filled-in usingthe Klenow fragment of DNA polymerase I. This blunt-end fragment wasre-circularized to give pALK19. The gene and promoter were excised frompALK19 as a SpeI/XbaI fragment and cloned into pLSB3 (Example 2)digested with SpeI. This step destroys the XbaI site at the end of theccr gene. The resulting plasmid is designated pALK21. Plasmid pALK21,therefore, contains the ccr gene under P_(actI) with unique NdeI andXbaI sites situated for the introduction of hybrid PKS genes downstreamof the ptr promoter. pALK21 is apramycin resistant and contains an oriTfor conjugal transfer of DNA.

EXAMPLE 19 Generation of a S. Spinosa Strain Harboring a HybridPolyketide Synthase Comprising the Rapamycin Module 2 AT in Place of theSpinosyn Module 3 AT

See FIG. 11. Saccharopolyspora spinosa NRRL 18538 was transformed byconjugation (Matsushima et al., 1994) from E. coli S17-1 (Simon et al.,1983) with pALK39. Transformants were selected for resistance toapramycin. A number of transformants were screened by Southern blotanalysis. A single transformant, displaying the correct hybridizationpattern to show that the plasmid had integrated into the chromosome byhomologous recombination, was designated strain S. spinosa 7D23.

Examples 20-21 hereinafter illustrate the use of S. spinosa 7D23 toproduce novel spinosyns.

EXAMPLE 20 Production and Isolation of 16-Desmethyl Spinosyns(Production of Spinosyn F, Spinosyn F Pseudoaglycone, and Compounds 21and 22)

16-Desmethyl spinosyn A has previously been identified as one of thefamily of spinosyns produced by a number of S. spinosa strains, anddesignated spinosyn F (U.S. Pat. No. 6,274,350 B1). Here we demonstratedproduction of spinosyn F and 16-desmethyl spinosyn D (Compound 21) fromthe engineered hybrid pathway of S. spinosa 7D23.

S. spinosa 7D23 was used to inoculate CSM medium (Hosted and Baltz 1996;U.S. Pat. No. 5,362,634). This pre-culture was grown in a 250 mL flaskwith a 30 cm spring to aid aeration, shaken at 250 rpm with a two-inchthrow, at 30° C. with 75% relative humidity for 3 days. It was then usedto inoculate vegetative medium, 3×30 mL cultures in 250 mL flasks(Strobel and Nakatsukasa 1993; U.S. Pat. No. 5,362,634) at 5% v/v, whichwas cultured under the same conditions for a further 2 days. Thevegetative culture was used to inoculate production medium (Strobel andNakatsukasa 1993) at 5% v/v, 30×30 mL cultures in 250 mL flasks grownunder the conditions described above. The production culture of S.spinosa 7D23 was grown for 10 days.

For the identification of metabolites, a 1 mL aliquot of fermentationbroth was analyzed by LC-MS as described in Example 5. By comparison toauthentic standards, it was clear that spinosyn F, 16-desmethyl spinosynD, and their corresponding pseudoaglycones were present (Table 12).

TABLE 12 Retention Key mass spectral Yield Compound time (min) data(m/z) (mg/l) spinosyn F 21.1 718.5 [M + H]⁺; 142.4 102 16-desmethylspinosyn D 23.4 732.5 [M + H]⁺; 142.3 38 (Compound 21) spinosyn F 17-23.1 599.3 [M + Na]⁺ 125 pseudoaglycone 16-desmethyl spinosyn D 24.6613.3 [M + Na]⁺ 42 17-pseudoaglycone (Compound 22)

The remaining fermentation broth was clarified by centrifugation. Thecells were extracted twice with an equal volume of methanol. Thesupernatant (780 mL) was adjusted to pH˜10 with 5 N NaOH and twiceextracted with ethyl acetate (3×500 mL). The methanol and ethyl acetateextracts were combined and the solvents removed in vacuo to give an oilyresidue. The residue was dissolved into ethyl acetate (250 mL) andextracted with 50 mM tartaric acid (3×200 mL). The combined tartaricacid extracts were adjusted to pH-10 with 5 N NaOH and extracted withethyl acetate (3×300 mL). The extracts were combined and the solventremoved in vacuo to yield a brown oil (200 mg). The oil was dissolvedinto methanol (1 mL) and half of this was initially chromatographed asdescribed in Example 8.

The major fractions from the initial separation that contained16-desmethyl spinosyn D and spinosyn F were combined and the solventremoved in vacuo. The residues were chromatographed over the samecolumn, eluting with a gradient as described below at a flow rate of 21mL/minute.

-   -   T=0 minute, 35% B; T=35, 55% B; T=45, 55% B.

Fractions were collected every 30 seconds. Fractions containing either16-desmethyl spinosyn D or spinosyn F were combined separately. Each ofthe combined set of fractions was then worked up as follows: theacetonitrile removed in vacuo, and the sample concentrated using aC₁₈-BondElute cartridge (200 mg). The sample was applied under gravity,washed with water (10 mL) eluted with methanol (2×10 mL), and thesolvent was removed in vacuo.

16-Desmethyl spinosyn D (Compound 21) has the following characteristics.

-   -   Isolated yield: 4.8 mg    -   Molecular weight: 731    -   Molecular formula: C₄₁H₆₅NO₁₀    -   UV (by diode array detection during HPLC analysis): 244 nm    -   Electrospray MS: m/z for MH⁺=732.5; forosamine sugar fragment        ion at m/z=142.4.    -   Accurate FT-ICR-MS: m/z for [MH]⁺=732.4689 (requires 732.4681).

Table 13 summarizes the ¹H and ¹³C NMR chemical shift data for16-desmethyl spinosyn D in CDCl₃.

TABLE 13 Position ¹H ¹³C  1 — 172.3   2a 2.39 33.9  2b 3.09 —  3 2.9747.7  4 3.37 42.1  5 5.49 122.0   6 — 136.3   6-Me 1.73 20.7  7 2.1944.5  8a 1.41 34.8  8b 1.93 —  9 4.29 75.7 10a 1.35 37.7 10b 2.26 — 111.01 45.6 12 2.76 49.0 13 6.75 148.7  14 — 145.1  15 — 198.0  16a 2.4745.1 16b 3.17 — 17 4.05 74.4 18a 1.56 33.7 18b 1.56 — 19a 1.17 21.5 19b1.67 21.5 20a 1.51 29.7 20b 1.51 — 21 4.65 76.9 22a 1.49 28.2 22b 1.49 —23 0.82  9.3  1′ 4.85 95.5  2′ 3.52 77.6  3′ 3.46 81.1  4′ 3.12 82.3  5′3.54 67.9  6′ 1.28 17.8  2′-OMe 3.51 59.1  3′-OMe 3.50 57.7  4′-OMe 3.5661.0  1″ 4.60 98.9  2″a 1.67 30.0  2″b 1.99 —  3″a 1.70 20.5  3″b 2.16 — 4″ 3.10 64.6  5″ 3.73 70.7  6″ 1.47 18.6  4″-NMe₂ Not seen* Not seen*Chemical shifts referenced to the proton of 7.26 ppm. *Not seen due totrace acid causing protonation of amine group.

Spinosyn F has the following characteristics.

-   -   Isolated yield: 5.5 mg    -   Molecular weight: 717    -   Molecular formula: C₄₀H₆₃NO₁₀    -   UV (by diode array detection during HPLC analysis): 244 nm    -   Electrospray MS: m/z for MH⁺=718.5; forosamine sugar fragment        ion at m/z=142.4.    -   Accurate FT-ICR-MS: m/z for [MH]⁺=718.4534 (requires 718.4525).

The NMR data accumulated for this compound were in agreement with itsproposed identity and with published data (U.S. Pat. No. 5,362,634).

EXAMPLE 21 Production and Isolation of 16-Desmethyl Spinosyn D17-Pseudoaglycone (Compound 22)

16-Desmethyl spinosyn A 17-pseudoaglycone was previously identified andis known as spinosyn F 17-pseudoaglycone. It is found as one of theminor members of the family of spinosyns produced by S. spinosa strains(U.S. Pat. No. 6,274,350 B1). Described below is the process for theproduction of spinosyn F 17-pseudoaglycone and 16-desmethyl spinosyn D17-pseudoaglycone from the engineered hybrid pathway of S. spinosa 7D23.

Frozen vegetative stocks of S. spinosa 7D23 were used to inoculateprimary vegetative pre-cultures of S. spinosa 7D23 in CSM (50 mLincubated in a 250 mL Erlenmeyer flask with spring). Secondarypre-cultures in vegetative medium (250 mL incubated in a 2 L Erlenmeyerflask with spring) were prepared and incubated as described in Example8.

Four liters of production medium were prepared, as in Example 6, withthe addition of 0.01% v/v Pluronic L-0101 (BASF) antifoam. Productionmedium was inoculated with the secondary pre-culture at 5% v/v and wasallowed to ferment in a 7 L stirred bioreactor for 7-10 days at atemperature of 30° C. Airflow was set at 0.75 vvm, and impeller tipspeed was controlled between 0.68 and 1.1 ms⁻¹ in order to maintaindissolved oxygen tension at or above 30% of air saturation.

For the identification of metabolites, a 1 mL aliquot of fermentationbroth was analyzed by LC-MS as described in Example 5. By comparison toauthentic standards, it was clear that spinosyn F, 16-desmethyl spinosynD and their corresponding pseudoagylcones were present (Table 14).

TABLE 14 Retention time Key mass spectral Compound (minute) data (m/z)Spinosyn F 21.4 718.4 [M + H]⁺; 142.4 16-desmethyl spinosyn D 23.6 732.5[M + H]⁺; 142.3 (Compound 21) Spinosyn F 17- 23.2 599.3 [M + Na]⁺pseudoagylcone 16-desmethyl spinosyn 24.3 613.3 [M + Na]⁺ D17-pseudoagylcone (Compound 22)

The remaining fermentation broth was clarified by centrifugation. Thecells were twice extracted with an equal volume of methanol. Thesupernatant (3.2 L) was extracted with ethyl acetate (2×1.5 L). Themethanol and ethyl acetate extracts were combined and the solventremoved in vacuo. The residual oil was dissolved into ethyl acetate (1L) and washed with 50 mM tartaric acid (3×500 mL). The remaining ethylacetate solution was evaporated in vacuo. The resulting oil waschromatographed over flash silica gel (6×13 cm) eluting with 5% methanolin chloroform. Fractions of 10 mL volume were collected. The fractionscontaining pseudoagylcones were combined and the solvent removed invacuo to yield a brown oil. The brown oil was dissolved into methanol(1.5 mL) and chromatographed over base-deactivated reversed-phase silicagel as described in Example 8. Fractions were collected every 30 secondsand those containing the relevant products were combined, theacetonitrile was removed in vacuo and the sample concentrated usingC₁₈-BondElute cartridges (200 mg). The sample was applied under gravity,washed with water (10 mL), eluted with methanol (2×10 mL), and thesolvent removed in vacuo.

16-desmethyl spinosyn D 17-pseudoagylcone (Compound 22) had thefollowing characteristics:

-   -   Isolated yield: 3.3 mg    -   Molecular weight: 590    -   Molecular formula: C₃₃H₅₀O₉    -   UV (by diode array detection during HPLC-MS analysis): 240 nm    -   Electrospray MS: m/z for [M+Na]⁺=613.3    -   Accurate FT-ICR-MS: m/z for [M+H]⁺=591.3517 (requires:        591.3528).

Table 15 summarizes the ¹H and ¹³C NMR spectral data for 16-desmethylspinosyn D 17-pseudoagylcone in CDCl₃.

TABLE 15 Position ¹H ¹³C  1 — 172.5   2a 2.40 34.0  2b 3.13 —  3 2.9747.7  4 3.37 42.4  5 5.49 122.1   6 — 136.3   6-Me 1.73 20.7  7 2.1944.5  8a 1.42 34.5  8b 1.93 —  9 4.30 75.6 10a 1.36 37.7 10b 2.28 — 111.03 45.6 12 2.76 48.9 13 6.77 148.0  14 — 145.1  15 — 197.8  16a 2.5647.5 16b 3.21 — 17 4.18 68.2 18a 1.58 35.7 18b 1.58 — 19a 1.20 21.1 19b1.60 — 20a 1.45 30.5 20b 1.56 — 21 4.68 76.3 22a 1.50 28.0 22b 1.50 — 230.83  9.4  1′ 4.86 95.5  2′ 3.51 77.7  3′ 3.46 81.1  4′ 3.12 82.3  5′3.54 67.9  6′ 1.28 17.8  2′-OMe 3.51 59.0  3′-OMe 3.51 57.7  4′-OMe 3.5660.9

b. Hybrid Spinosyn PKS Using 5 AT Module of the Tylosin PKS in Place ofSpinosyn Module 3 AT

In an analogous way, a hybrid spinosyn PKS was generated in which themethyl malonyl-CoA specific AT domain of module 3 was replaced by theethyl malonyl-CoA specific AT domain of tylosin module 5 (SEQ ID NO:26).The producing strain was designated S. spinosa 36P4. Ethyl malonyl-CoAwas not expected to be abundant in S. spinosa, so the S. cinnamonensisgene encoding crotonyl-CoA reductase was expressed in the same cell,under control of the actI promoter. This should significantly increasethe intracellular pool of butyryl-CoA, which is a substrate for shortchain fatty acid carboxylases that can provide ethyl malonyl-CoA. ThePKS of S. spinosa 36P4 contained spnA, spnB and a truncated spnC underthe native spnA promoter, followed by the plasmid DNA including theapramycin resistance marker. The S. cinnamonensis crotonyl-CoA reductaseunder the actI promoter, and the actII-ORF4 activator, were also withinthe plasmid sequence. This was followed by the hybrid spnC (spnC*) geneunder control of the promoter for resistance to pristinamycin. Oneskilled in the art will appreciate that the heterologous promoters couldbe swapped around, or indeed that a number of other promoters could bechosen.

Strain S. spinosa 36P4 produced minor components that had the UVabsorbance, chromatographic properties, masses and fragmentationpatterns consistent with the expected 16-desmethyl-16-ethyl spinosyns Aand D. The major products from S. spinosa 36P4 were spinosyns A and D.Surprisingly, the predominant novel fermentation products of strain 36P4were 21-desethyl-21-n-propyl spinosyn A and 6-ethyl spinosyn A.21-desethyl-21-n-propyl spinosyn A was produced at levels within 10% ofthat of spinosyn D. These compounds were isolated and fullycharacterized by MS and NMR. We suggest that they were made due to anincrease in the intracellular concentration of ethyl malonyl-CoA, whichresulted from the introduction of the crotonyl-CoA reductase gene. Thelow specificity of both the loading AT and the module 8 AT allowed thisnovel substrate to be incorporated. 6-Ethyl-21-desethyl-21-n-propylspinosyn A was also made by S. spinosa 36P4, as a minor factor. Thesethree products each have a methyl group at C16, which implies that thetylosin module 5 AT, in this system, predominantly incorporatedmethylmalonyl-CoA. It is, therefore, expected that a non-engineeredspinosyn PKS (with the native AT3) would produce the 21-n-propyl and6-ethyl spinosyn compounds in the presence of the ccr gene.

EXAMPLE 22 Construction of a Vector that can be Used to Engineer theSpinosyn Biosynthetic Pathway to Produce 16-Desmethyl-16-Ethyl Spinosyns

See FIG. 10. Plasmid pTB4 is a pUC18-based plasmid containing a BamHIfragment of the tylosin PKS that includes most of the tylosin module 4.The insert is from the BamHI site between by 24125 and 24130 of thedeposited sequence tylG.embo, accession number U78289 (at the beginningof KS4) and the BamHI site between by 31597 and 31612 (at the beginningof KS5).

The tylosin module 5 AT was amplified by PCR using the template pTB4 andthe primers AK1 (SEQ ID NO:24) and AK2 (SEQ ID NO:25). The primer AK1introduces an MscI site (bp 3-8 of SEQ ID NO:24) at the beginning of theAT domain and primer AK2 introduces an AvrII site (bp 3-8 of SEQ IDNO:25) at the end of the AT domain. The PCR reaction was carried outunder standard conditions using Pwo thermostable DNA polymerase. Thefragment was phosphorylated with T4 polynucleotide kinase and clonedinto commercially available pUC18 digested with SmaI anddephosphorylated. Insert-containing plasmids were analyzed for theorientation of the insert and sequenced. One plasmid containing thecorrect sequence was identified and designated pALK17. It contains thePCR fragment in the orientation that places the MscI site adjacent tothe HindIII site of the polylinker. The tyl AT5 was excised from pALK17as an MscI/AvrII fragment and cloned into pALK26 digested with MscI andAvrII to give pALK27. The 407 bp MscI fragment that is missing from thisconstruct was excised from pALK24 (Example 16) and ligated intodephosphorylated, MscI-digested pALK27. A single clone containing theinsert in the correct orientation was designated pALK31. pALK31 containsthe required fragment to introduce the tyl AT5 swap into module 3 of S.spinosa, with an NdeI site at the start codon and a XbaI site justdownstream of the polyketide synthase sequence. This fragment wasexcised as an NdeI/XbaI fragment and cloned into pALK21 to give pALK36.This places the new module 3 hybrid fragment under P_(ptr) in a vectorthat co-expresses the ccr from the actI promoter, and can be transferredinto S. spinosa by conjugation and selected for apramycin resistance.

EXAMPLE 23 Generation of a S. Spinosa Strain Harboring a HybridPolyketide Synthase Comprising the Tylosin Module 5 AT in Place of theSpinosyn Module 3 AT, and Providing the Appropriate Ethyl Malonyl-CoACo-Substrate

See FIG. 12. Saccharopolyspora spinosa NRRL 18538 was transformed withpALK36. Transformants were selected for resistance to apramycin andscreened by Southern blot analysis. A single transformant was designatedstrain S. spinosa 36P4.

EXAMPLE 24 Production and Isolation of Compounds from S. Spinosa 36P4

Frozen vegetative stocks of S. spinosa 36P4 were inoculated into primaryvegetative pre-cultures of S. spinosa 36P4 in CSM (50 mL incubated in a250 mL Erlenmeyer flask with spring). Secondary pre-cultures invegetative medium (250 mL incubated in a 2 L Erlenmeyer flask withspring) were prepared and incubated as described in Example 8.

Fourteen liters of production medium were prepared, as in Example 6,with the addition of 0.01% v/v Pluronic L-0101 (BASF) antifoam.Production medium was inoculated with the secondary pre-culture at 5%v/v and was allowed to ferment in a 20 L stirred bioreactor for 7-10days under conditions described in Example 8.

For the identification of metabolites, a 1 mL aliquot of fermentationbroth was analyzed by LC-MS as described in Example 5. By comparison toauthentic standards, and to a fermentation extract from strain S.spinosa NRRL 18538, the presence of new spinosyn metabolites wasverified (Table 16). The major new component eluted with a similar—butdifferent—retention time to spinosyn D and had an identical mass; thiscompound is identified below as 21-desethyl-21-n-propyl spinosyn A. Thesecond significant new component eluted later and had a mass 14 unitshigher than spinosyn D; this compound is identified below as 6-ethylspinosyn A. The third significant peak eluted later still and had a mass28 units higher than spinosyn D; this compound is believed to be6-ethyl-21-desethyl-21-n-propyl spinosyn A. The mass spectra of all ofthese compounds displayed a [M+H]⁺ ion plus the forosamine fragment. Inaddition, several other new components were clearly present but werepresent in minor quantities. One of these new components eluted afterthe first major new component and had an identical mass to spinosyn D;this compound was probably 16-desmethyl-16-ethyl spinosyn A. Other minornew components displayed a [M+H]⁺ ion 14 mass units higher than spinosynD and a mass consistent with the forosamine fragment. One of these newminor components may be 16-desmethyl-16-ethyl spinosyn D.

TABLE 16 Compound Retention No. (See time Key mass spectral CompoundTable 3) (minutes) data (m/z) 21-desethyl-21-n- 23 25.4 746.5 [M + H]⁺;142.3 propyl spinosyn A 6-ethyl spinosyn A 24 27.1 760.5 [M + H]⁺; 142.46-ethyl-21-desethyl- 25 29.1 774.5 [M + H]⁺; 142.3 21-n-propyl spinosynA 16-desmethyl-16-ethyl 26 26.4 746.5 [M + H]⁺; 142.3 spinosyn Aputative 16-desmethyl- 27 26.7 760.4 [M + H]⁺; 142.4 16-ethyl spinosyn Dputative 16-desmethyl- 27 27.3 760.4 [M + H]⁺; 142.4 16-ethyl spinosyn Dputative 16-desmethyl- 27 27.5 760.5 [M + H]⁺; 142.3 16-ethyl spinosyn D

The remaining fermentation broth (12 L) was clarified by centrifugationand extracted as described in Example 8. The residue was dissolved intomethanol (10 mL), water (2 mL) and formic acid (100 μl). The wholesample was filtered and applied under gravity to a C₁₈-BondElute SPEcartridge (70 g, 150 mL). The cartridge was then developed with anincreasing 10%-stepwise gradient of acetonitrile in water (100 mL eachstep) containing formic acid at 0.1% using a FlashMaster Personal system(Jones Chromatography, Wales UK). The column was finally washed withmethanol (2×100 mL). Fractions containing spinosyn-like molecules with amass of 746 amu or greater were combined and the solvents removed invacuo. The residual oil (3 mL) was dissolved in methanol (1.5 mL). Thissample was initially chromatographed in two equal portions as describedin Example 8.

The fractions from the initial pair of separations that contained6-ethyl spinosyn A and 21-desethyl-21-n-propyl spinosyn D (m/z=760) werecombined and the solvent removed in vacuo. The residue was dissolved inmethanol (1 mL) and chromatographed over reversed-phase silica gel(Hypersil C₁₈-BDS, 5 μm; 21×250 mm) eluting with a gradient as describedbelow, at a flow rate of 21 mL/minute.

-   -   T=0 minute, 40% B; T=80, 50% B.

Fractions were collected every 30 seconds. Fractions containingpredominantly 6-ethyl spinosyn A were combined, the acetonitrile removedin vacuo, and the sample concentrated using a C₁₈-BondElute cartridge(200 mg), washed with water (10 mL) and eluted with methanol (2×10 mL),and the solvent removed in vacuo. Fractions from the initial pair ofseparations that contained mainly 21-desethyl-21-n-propyl spinosyn A(m/z=746) were combined and the solvent removed in vacuo. The residueswere dissolved in methanol:water (7:3, 1 mL) and chromatographed overthe same column eluting with the following gradient at 21 mL/minute.

-   -   T=0 minute, 40% B; T=45, 80% B.

Fractions were collected every 30 seconds. Fractions containing only21-desethyl-21-n-propyl spinosyn A were combined. Samples containing amixture of this compound with spinosyn D were combined separately, thesolvent removed in vacuo, and the residue chromatographed once again asdescribed above. The fractions containing only 21-desethyl-21-n-propylspinosyn A were then combined with those from the first run. Thefractions containing a mixture of the two compounds were combined andanother round of chromatography performed.

The fractions from the three runs that contained only21-desethyl-21-n-propyl spinosyn A were combined, the acetonitrileremoved in vacuo and the sample concentrated using a C₁₈-BondElutecartridge (200 mg). The sample was applied under gravity, washed withwater (10 mL), eluted with methanol (2×10 mL), and the solvent removedin vacuo.

6-Ethyl spinosyn A (Compound 24) has the following characteristics.

-   -   Isolated yield: 4.8 mg    -   Molecular weight: 759    -   Molecular formula: C₄₃H₆₉NO₁₀    -   UV (by diode array detection during HPLC analysis): 244 nm

Electrospray MS: m/z for MH⁺=760.5; forosamine sugar fragment ion atm/z=142.4.

-   -   Accurate FT-ICR-MS: m/z for [MNa]⁺=782.4818 (requires 782.4814).

Table 17 summarizes the ¹H and ¹³C NMR chemical shift data for 6-ethylspinosyn A in CDCl₃.

TABLE 17 Position ¹H ¹³C  1 — 172.6   2a 2.42 34.0  2b 3.13 —  3 2.9747.9  4 3.44 41.9  5 5.46 120.3   6 — 141.8   7 2.23 44.4  8a 1.42 34.5 8b 1.95 —  9 4.30 75.8 10a 1.35 37.7 10b 2.26 — 11 1.00 45.9 12 2.7749.1 13 6.76 147.7  14 — 144.4  15 — 202.9  16 3.28 47.7 17 3.63 80.618a 1.52 34.3 18b 1.52 — 19a 1.20 21.7 19b 1.77 — 20a 1.52 30.0 20b 1.52— 21 4.67 76.6 22a 1.49 28.4 22b 1.49 — 23 0.82  9.3 24a 2.05 27.4 24b2.05 — 25 1.03 12.6 26 1.17 16.1  1′ 4.85 95.5  2′ 3.50 77.7  3′ 3.4781.0  4′ 3.12 82.3  5′ 3.55 67.9  6′ 1.28 17.8  2′-OMe 3.50 59.0  3′-OMe3.50 57.7  4′-OMe 3.56 60.9  1″ 4.43 103.4   2″a 1.50 30.8  2″b 1.99 — 3″a 1.47 18.6  3″b 1.88 —  4″ 2.28 64.9  5″ 3.49 73.4  6″ 1.28 19.0 4″-NMe₂ 2.28 40.6 Chemical shifts referenced to the proton of CHCl₃ at7.26 ppm.

21-desethyl-21-n-propyl spinosyn D (Compound 28) has the followingcharacteristics.

-   -   Isolated yield: ˜1 mg    -   Molecular weight: 759    -   Molecular formula: C₄₃H₆₉NO₁₀

This compound was present as the minor component in a 4:1 mixture with6-ethyl spinosyn A. The accumulated UV and MS data for these twocompounds are indistinguishable. Using NMR methods, the 21-n-propyl spinsystem could be assigned from correlations observed in the COSY spectrumof the mixture. Methyl H24 (δ_(H) 0.87, dd, 7.3 Hz, 7.3 Hz) wascorrelated to the methylene H23 (δ_(H) 1.23, m). H23 was correlated tothe methylene H22 (δ_(H) 1.43, m) that in turn was correlated to H21(δ_(H) 4.73, m). The H21 resonance was visible as an isolated multipletin the ¹H NMR spectrum of the mixture.

21-desethyl-21-n-propyl spinosyn A (Compound 23) has the followingcharacteristics.

-   -   Isolated yield: 5.1 mg    -   Molecular weight: 745    -   Molecular formula: C₄₂H₆₇NO₁₀    -   UV (by diode array detection during HPLC analysis): 244 nm    -   Electrospray MS: m/z for MH⁺=746.5; forosamine sugar fragment        ion at m/z=142.4.

The accumulated NMR data for this compound were identical to thosedescribed for this compound in Example 15.

Hybrid PKS genes constructed by the replacement of other AT(acyltransferase) domains within spn extender modules can be used toproduce novel spinosyns with altered side chains at other positions onthe polyketide. For example, in analogous methods to those describedabove, hybrid polyketide synthases can be constructed to yield spinosynsin which C18 or C20 bears a side chain other than a hydrogen (generallya methyl or ethyl). The native AT domains that incorporate predominantlymethylmalonyl-CoA (such as spn AT3) can be replaced by heterologousdomains that preferentially incorporate malonyl-CoA (such as rapamycinAT2) or ethylmalonyl-CoA (such as tylosin AT5). However, one skilled inthe art will recognize that donor domains or modules for these hybridpolyketide synthases could be acquired from a variety of Type Ipolyketide synthase clusters and that this is not restricted in any wayto domains or modules from erythromycin, avermectin, rapamycin andtylosin biosynthesis.

It is also anticipated that a combination of manipulations should leadto productive biosynthetic pathways, and spinosyns with two or moreregions of novelty.

Additional biosynthetic genes may be required to provide an adequatesupply of a precursor that is not normally incorporated into spinosyns,such as a ccr gene to increase ethylmalonyl-CoA supply. This geneticmodification can also lead to the production of novel spinosyns byproviding an unnatural precursor that is incorporated at other spn ATdomains. The replacement of other spn AT domains could generate hybridPKS genes that lead to the synthesis of spinosyns with an ethyl group atC6, or methyl or ethyl side chains at C18 or C20. The spn PKS domainsresponsible for the degree of modification of each beta-keto group (KR,DH or ER) might also be replaced by heterologous domains to generatehybrid PKS genes that result in spinosyns with different saturatedbonds, hydroxyl groups or double bonds.

In summary, we have demonstrated that the hybrid spinosyn PKS genesclaimed herein are useful for the production of novel, insecticidallyactive spinosyns. The hybrid genes are derived from the spn PKS genescombined with a portion or portions of other Type I PKS genes. Thestrategies described in WO 98/01546 “Polyketides and their synthesis”were used to select the sites where the DNAs are spliced together. Thehybrid genes can be operably linked to a heterologous promoter such asthat from the actinorhodin biosynthetic gene actI (along with theactII-ORF4 gene encoding its cognate activator, see WO 98/01546), orfrom the pristinamycin resistance gene ptr (Blanc et al., 1995). Thehybrid PKS genes are expressed in an organism that also contains thenon-PKS functions required to produce a biologically active spinosyn.The modified strains provided by the invention may be cultivated toprovide spinosyns using conventional protocols such as those disclosedin U.S. Pat. No. 5,362,634.

It is contemplated that the hybrid spinosyn PKSs of the invention can beexpressed not only in Saccharopolyspora spinosa, but also in other hostorganisms, for example Saccharopolyspora erythaea, to produceinsecticidally active spinosyns. Other prokaryotic cells belonging tothe group of actinomycetes, preferably the group of streptomycetes, arealso suitable host organisms. Streptomyces albus is a specific example.

Pesticide Activity of New Spinosyns

The compounds claimed herein are useful for the control of insects andmites. Included are all isomers of the compounds, and any acid additionsalts of the compounds and their isomers. Also included aresemi-synthetic derivatives made by the methods described in U.S. Pat.No. 6,001,981 to prepare other modified spinosyns.

The compounds show activity against a number of insects and mites. Morespecifically, the compounds show activity against members of the insectorder Lepidoptera such as the beet armyworm, tobacco budworm, codlingmoth and cabbage looper. They also show activity against members of theorder Coleoptera (the beetles and weevils) and Diptera (the true flies).The compounds also show activity against members of the order Hempitera(true bugs), Homoptera (aphids and hoppers), Thysanoptera (thrips),Orthoptera (cockroaches), Siphonaptera (fleas), Isoptera (termites), andmembers of the Hymenoptera order Formicidae (ants). The compounds alsoshow activity against the two-spotted spider mite, which is a member ofthe Arachnid order Acarina.

A further aspect of the present invention is directed to methods forinhibiting an insect or mite. In one preferred embodiment, the presentinvention is directed to a method for inhibiting a susceptible insectthat comprises applying to a plant an effective insect-inactivatingamount of compound in accordance with the present invention. The claimedcompounds are applied in the form of compositions, which are also a partof this invention. These compositions comprise an insect- ormite-inactivating amount of compound in an inert carrier. The activecomponent may be present as a single claimed compound, a mixture of twoor more compounds or a mixture of any of the compounds together with thedried portion of the fermentation medium in which it is produced.Compositions are prepared according to the procedures and formulas thatare conventional in the agricultural or pest control art, but that arenovel and important because of the presence of one or more of thecompounds of this invention. The compositions may be concentratedformulations, which are dispersed in water or may be in the form of adust, bait or granular formulation used without further treatment.

The action of the compositions according to the invention can bebroadened considerably by adding other, for example insecticidally,acaricidally, and/or nematocidally active, ingredients. For example, oneor more of the following compounds can suitably be combined with thecompounds of the invention: organophosphorus compounds, carbamates,pyrethroids, acylureas, other types of insect growth regulators andinsect hormone analogs, neonicotinoids and other nicotinics, macrolidesand other insecticidal, acaricidal, mollscicial and nematocidalcompounds or actives. WO 00/56156 on “Synergistic Insecticide Mixtures”discloses use of certain previously known spinosyn compounds incombination with agonists or antagonists of nicotinic acetylcholinereceptors to control animal pests. WO 00/35282 on “Combination of ActiveIngredients” discloses use of spinosad in combination with afungicidally active compound. WO 00/35286 on “Combinations of ActiveIngredients” discloses use of a combination of spinosad with othercompounds to control animal pests and fungi. WO 99/60856 on “Use ofSpinosyns as Soil Insecticides” discloses use of certain previouslyknown spinosyns for treating seeds and for application to plants via thesoil or by irrigation to control insects. WO 99/33343 on “Use ofMacrolides in Pest Control” discloses use of spinosyns to control pestsin transgenic crops, use of spinosyns to protect plant propagationmaterial and plant organs formed at a later time from attack by pests,and use of spinosyns to control wood pests and mollusks. The compoundsof Formula I can also be used for these purposes.

The compounds of the present invention are also useful for the treatmentof animals to control arthropods, i.e., insects and arachnids includingvarious flies and fly larvae, fleas, lice, mites, and ticks, which arepests on animals. Techniques for delivering ectoparasiticides are wellknown to those skilled in the art. In general, a present compound isapplied to the exterior surface of an animal by sprays, dips or dusts.The compounds can also be delivered to animals using ear tags, adelivery method disclosed in U.S. Pat. No. 4,265,876.

In yet another embodiment, the compounds can be used to control insectsand arachnids that are pests in the feces of cattle and other animals.In this embodiment, the compounds are administered orally and thecompounds travel through the intestinal tract and emerge in the feces.Control of pests in the feces indirectly protects the animals from thepests.

The compounds of the invention are also useful as human pharmaceuticalsto control parasites, for example, lice. The compounds can be used, forexample, in the formulations for controlling lice that are disclosed inWO 00/01347.

EXAMPLE 25 Demonstration that Novel Purified Spinosyns are Insecticidal

Biological activity of the compounds of the invention was shown by atopical assay in which the compound was applied to laboratory-rearedlarvae (mean weight 22 mg) at the rate of 1 microg/larva. Each compoundwas applied, in an acetone solution (1 mg/mL), along the dorsum of sixtobacco budworm (Heliothis virescens) larvae and six beet armyworm(Spodoptera exigua) larvae. Treated larvae were then held for two daysat 21° C., 60% RH in six-well plastic culture plates. Larvae were eachsupplied with a 1 cm³ of agar-based Lepidoptera diet for sustenanceduring the two-day post-exposure interval. Percent mortality wasdetermined at the end of a two-day period (Table 18).

TABLE 18 Tobacco Budworm Beet Armyworm Compound Rate Mor- Rate Mor- No.(See (micro/ tality (microg/ tality Compound Table 3) larva) (%) larva)(%) solvent only 0 0 0 0 21-cyclopropyl 1 1 100 1 33 21-cyclobutyl 3 183 1 83 21-cyclobutyl, 4 1 100 1 83 6-methyl 21-cyclobutyl, 8 1 100 1 835,6-dihydro 21-isopropyl 9 1 100 1 100 21-n-propyl 23 1 100 1 100

The compounds of formula (I) can be used as intermediates in theprocesses disclosed in U.S. Pat. No. 6,001,981 to produce semi-syntheticspinosyn analogues, which are also expected to have insecticidalactivity.

The US patents and patent applications cited hereinabove are herebyincorporated by reference.

REFERENCES

-   1) Bierman M., R. Logan, K. O'Brien, E. T. Seno, R. Nagaraja Rao,    and B. E. Schoner (1992). “Plasmid cloning vectors for the conjugal    transfer of DNA from Escherichia coli to Streptomyces spp.” Gene    116:43-49.-   2) Bisang C., P. F. Long, J. Cortés, J. Westcott, J. Crosby, A. L.    Matharu, R. J. Cox, T. J. Simpson, J. Staunton, and P. F. Leadlay    (1999)/“A chain initiation factor common to both modular and    aromatic polyketide synthases.” Nature 401:502-505.-   3) Blanc V., K. Salah-Bey, M. Folcher, and C. J. Thompson (1995).    “Molecular characterization and transcriptional analysis of a    multidrug resistance gene cloned from the pristinamycin-producing    organism, Streptomyces pristinaespiralis.” Mol. Microbiol.    17:989-999.-   4) Broughton M. C., M. L. B. Huber, L. C. Creemer, H. A. Kirst,    and J. R. Turner (1991). “Biosynthesis of the macrolide insecticidal    compound A83543 by Saccharopolyspora spinosa.” Proceedings of Amer.    Soc. Microbiol., Washington D.C.-   5) Donadio S., M. J. Stayer, J. B. McAlpine, S. J. Swanson, and L.    Katz (1991). “Modular organization of genes required for complex    polyketide biosynthesis.” Science 252:675-679.-   6) Donadio S. A., D. Stassi, J. B. McAlpine, M. J. Stayer, P. J.    Sheldon, M. Jackson, S. J. Swanson, E. Wendt-Pienkowski, Y. G.    Wang, B. Jarvis, C. R. Hutchinson, and L. Katz (1993). “Recent    developments in the genetics of erythromycin formation.” In    Industrial microorganisms: basic and applied molecular genetics.    (R. H. Baltz, G. D. Hegeman, and P. L. Skatrud, eds), pp. 257-265.    Amer. Soc. Microbiol., Washington D.C.-   7) Dutton C. J., S. P. Gibson, A. C. Goudie, K. S. Holdom, M. S.    Pacey, J. C. Ruddock, J. D. Bu'Lock, and M. K. Richards (1991).    “Novel avermectins produced by mutational biosynthesis.” J.    Antibiot. 44:357-365.-   8) Hosted T. J. and R. H. Baltz (1996). “Mutants of Streptomyces    roseosporus that express enhanced recombination within partially    homologous genes.” Microbiology 142:2803-2813.-   9) Hunziker D., T. W. Yu, C. R. Hutchinson, H. G. Floss, and C.    Khosla (1998). “Primer unit specificity in rifamycin biosynthesis    principally resides in the later stages of the biosynthetic    pathway.” J. Am. Chem. Soc. 12:1092-1093.-   10) Kirst H. A., K. H. Michel, J. W. Martin, L. C. Creemer, E. H.    Chio, R. C. Yao, W. M. Nakatsukasa, L. D. Boeck, J. L.    Occolowitz, J. W. Paschal, J. B. Deeter, N. D. Jones, and G. D.    Thompson (1991). “A83543A-D, unique fermentation-derived tetracyclic    macrolides.” Tetrahedron Letts 32:4839-4842.-   11) Marsden A. F. A., B. Wilkinson, J. Cortés, N. J. Dunster, J.    Staunton, and P. F. Leadlay (1998). “Engineering broader specificity    into an antibiotic-producing polyketide synthase.” Science    279:199-202.-   12) Matsushima P., M. C. Broughton, J. R. Turner, and R. H. Baltz    (1994). “Conjugal transfer of cosmid DNA from Escherichia coli to    Saccharopolyspora spinosa: effects of chromosomal insertions on    macrolide A83543 production.” Gene 146:39-45.-   13) Pacey M. S., J. P. Dirlam, R. W. Geldart, P. F.    Leadlay, H. A. I. McArthur, E. L. McCormick, R. A. Monday, T. N.    O'Connell, J. Staunton, and T. J. Winchester (1998). “Novel    erythromycins from a recombinant Saccharopolyspora erythraea strain    NRRL 2338 pIGI. Fermentation, isolation and biological activity.” J.    Antibiot. 51:1029-1034.-   14) Rowe C. J., J. Cortés, S. Gaisser, J. Staunton, and P. F.    Leadlay (1998). “Construction of new vectors for high-level    expression in actinomycetes.” Gene 216:215-223.-   15) Salah-Bey K., V. Blanc, and C. J. Thompson (1995).    “Stress-activated expression of a Streptomyces pristinaespiralis    multidrug resistance gene (ptr) in various Streptomyces spp. and    Escherichia coli.” Mol. Microbiol. 17:1001-1012.-   16) Simon R., U. Preifer, and A. Pühler (1983). “A broad host range    mobilization system for in vivo genetic engineering: transposon    mutagenesis in Gram negative bacteria.” Bio/Technology 1:784-791.-   17) Stassi D. L., S. J. Kakavas, K. A. Reynolds, G. Gunawardana, S.    Swanson, D. Zeidner, M. Jackson, H. Liu, A. Buko, and L. Katz    (1998). “Ethyl-substituted erythromycin derivatives produced by    directed metabolic engineering.” Proc. Natl. Acad. Sci. USA    95:7305-7309.-   18) Strobel R. J. and W. M. Nakatsukasa (1993). “Response surface    methods for optimizing Saccharopolyspora spinosa, a novel macrolide    producer.” J. Indust. Microbiol. 11:121-127.-   19) Waldron C., P. Matsushima, P. R. Rosteck, Jr., M. C.    Broughton, J. Turner, K. Madduri, K. P. Crawford, D. J. Merlo,    and R. H. Baltz (2001). “Cloning and analysis of the spinosad    biosynthetic gene cluster of Saccharopolyspora spinosa.” Chem. Biol.    8:487-499.

What is claimed is:
 1. A compound of the formula (I)

wherein R1 is hydrogen, methyl, or ethyl; R2 is hydrogen, methyl, orethyl; R3 is hydrogen,

R4 is methyl or ethyl, either of which may be substituted with one ormore groups selected from halo, hydroxy, C₁-C₄ alkyl, C₁-C₄ alkoxy,C₁-C₄ alkylthio, or cyano; or R4 is an alpha-branched C₃-C₅ alkyl group,C₃-C₈ cycloalkyl group, or C₃-C₈ cycloalkenyl group, any of which may besubstituted with one or more groups selected from halo, hydroxy, C₁-C₄alkyl, C₁-C₄ alkoxy, C₁-C₄ alkylthio, or cyano; or R4 is a 3-6 memberedheterocyclic group that contains O or S, that is saturated or fully orpartially unsaturated, and that may be substituted with one or moregroups selected from halo, hydroxy, C₁-C₄ alkyl, C₁-C₄ alkoxy, C₁-C₄alkylthio, or cyano; R5 is hydrogen or methyl; R6 is hydrogen or methyl;R7 is hydrogen or methyl; R8 is hydrogen, methyl, or ethyl; and R9 ishydrogen, methyl, or ethyl, and/or a 5,6-dihydro derivative of thecompound of formula (I), provided that the compound of formula (I) hasat least one of the following features: a) R4 is n-propyl, iso-propyl,cyclopropyl, methylcyclopropyl, sec-butyl, cyclobutyl, methylthio, orfuryl, or b) R1 or R2 is ethyl.
 2. The compound of claim 1, wherein R1is ethyl.
 3. The compound of claim 1, wherein R2 is ethyl.
 4. Thecompound of claim 1, wherein R4 is n-propyl, iso-propyl, cyclopropyl,methylcyclopropyl, sec-butyl, cyclobutyl, methylthio, or furyl.
 5. Acompound selected from the group consisting of:21-desethyl-21-cyclopropyl spinosyn A; 21-desethyl-21-cyclopropylspinosyn D; 21-desethyl-21-cyclobutyl spinosyn A;21-desethyl-21-cyclobutyl spinosyn D; 21-desethyl-21-methylthiomethylspinosyn A; 21-desethyl-21-methylthiomethyl spinosyn D;21-desethyl-21-cyanomethyl spinosyn A;5,6-dihydro-21-desethyl-21-cyclobutyl spinosyn A;21-desethyl-21-isopropyl spinosyn A; 21-desethyl-21-isopropyl spinosynD; 21-desethyl-21-sec-butyl spinosyn A; 21-desethyl-21-sec-butylspinosyn D; 21-desethyl-21-methylcyclopropyl spinosyn A;21-desethyl-21-methylcyclopropyl spinosyn D; 21-desethyl-21-(3-furyl)spinosyn A; 21-desethyl-21-(3-furyl) spinosyn D;21-desethyl-21-cyclopropyl spinosyn A 17-pseudoaglycone;21-desethyl-21-cyclopropyl spinosyn D 17-pseudoaglycone;21-desethyl-21-cyclobutyl spinosyn A 17-pseudoaglycone;21-desethyl-21-cyclobutyl spinosyn D 17-pseudoaglycone; 16-desmethylspinosyn D; 16-desmethyl spinosyn D 17-pseudoaglycone;21-desethyl-21-n-propyl spinosyn A; 6-ethyl spinosyn A;6-ethyl-21-desethyl-21-n-propyl spinosyn A; 16-desmethyl-16-ethylspinosyn A; 16-desmethyl-16-ethyl spinosyn D; and21-desethyl-21-n-propyl spinosyn D.
 6. A pesticide compositioncomprising an effective amount of the compound of claim 1 as activeingredient in combination with an appropriate diluent or carrier.
 7. Thepesticide composition of claim 6, wherein R1 is ethyl.
 8. The pesticidecomposition of claim 6, wherein R2 is ethyl.
 9. The pesticidecomposition of claim 6, wherein R4 is n-propyl, iso-propyl, cyclopropyl,methylcyclopropyl, sec-butyl, cyclobutyl, methylthio, or furyl.
 10. Apesticide composition comprising an effective amount of the compound ofclaim 5 as active ingredient in combination with an appropriate diluentor carrier.